Primers and Methods for Nucleic Acid Amplification

ABSTRACT

A primer and method for amplification of a target nucleic acid, the primer adapted to conform into a conformation that dissociates from a complementary strand of DNA duplex. The conformation may have a free energy with more favorable thermodynamics than a corresponding DNA duplex, such as a B-DNA duplex. The dissociation may occur during an extension step of an amplification method, such as polymerase chain reaction. The method can proceed isothermally, and the primers may include intrinsic fluorescence.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/579,486, filed Aug. 16, 2012, entitled “Primers and Methods forNucleic Acid Amplification,” which is a U.S. national phase filing under35 U.S.C. §371 of International Patent Application No.PCT/US2011/025411, filed Feb. 18, 2011, entitled “Primers and Methodsfor Nucleic Acid Amplification,” which claims priority to and thebenefit of the filing date of U.S. Provisional Patent Application No.61/338,475, entitled “Quadruplex Priming for Polymerase Chain Reaction,”filed on Feb. 19, 2010, the disclosures of which are hereby incorporatedby reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the polymerase chain reaction(PCR), and more specifically to novel primers used in PCR and a novelmethod for amplification of a target sequence in PCR.

BACKGROUND

This section is intended to introduce the reader to various aspects ofart that may be related to various aspects of the present invention,which are described and/or claimed below. This discussion is believed tobe helpful in providing the reader with background information tofacilitate a better understanding of various aspects of the presentinvention. Accordingly, it should be understood that these statementsare to be read in this light, and not as admissions of prior art.

Since its initial design by Kary Mullis in 1984, the polymerase chainreaction (PCR) has impacted nearly every field in molecular biology,genetics, and forensic science. PCR is a technique to amplify a singleor few copies of a particular nucleic acid sequence, e.g., DNA (thetarget DNA sequence), across several orders of magnitude, therebygenerating thousands to millions of copies of the sequence. PCR is themain tool presently used to amplify nucleic acid and study geneexpression.

PCR relies on “thermal cycling,” which includes cycles of repeatedheating and cooling of the DNA and other reaction components to causeDNA denaturation (i.e., separation of the double-stranded DNA into itssense and antisense strands) followed by enzymatic replication of theDNA. The other reaction components include short oligonucleotide DNAfragments known as “primers,” which contain sequences complementary toat least a portion of the target DNA sequence, and a DNA polymerase.These are components that facilitate selective and repeatedamplification of the target sequence. As PCR progresses, the DNAgenerated is itself used as a template for further replication insubsequent cycles, creating a chain reaction in which the target DNAsequence is exponentially amplified.

More specifically, the DNA polymerase used in PCR is thermostable (andthus avoids enzyme denaturation at high temperatures) and amplifiestarget DNA by in vitro enzymatic replication. One such thermostable DNApolymerase is Taq polymerase, an enzyme originally isolated from thebacterium Thermus aquaticus. The DNA polymerase enzymatically assemblesa new DNA strand from deoxynucleoside triphosphates (dNTPs) by using thedenatured single-stranded DNA as a template. As is known to those ofordinary skill in the art, a deoxynucleoside triphosphate isdeoxyribose) having three phosphate groups attached, and having one base(adenine, guanine, cytosine, thymine) attached. However, as used herein,it will be recognized by those of ordinary skill in the art that arsenicmay be substituted for phosphorous in the triphosphate back bone anydNTP. The initiation of DNA synthesis and the selectivity of PCR resultsfrom the use of primers that are complementary to the DNA regiontargeted for amplification under specific thermal cycling conditions.

Thus, a basic PCR set up includes multiple components. These include:(1) a DNA template that contains the target DNA region to be amplified;(2) primers that are complementary to the 3′ ends of each of the sensestrand and anti-sense strand of the target DNA; (3) a thermostable DNApolymerase such as Taq polymerase; and (4) dNTPs, the building blocksfrom which the DNA polymerases synthesizes a new DNA strand.Additionally, the reaction will generally include other components suchas a buffer solution providing a suitable chemical environment foroptimum activity and stability of the DNA polymerase, divalent cations(generally magnesium ions), and monovalent cation potassium ions (K⁺).

PCR is commonly carried out in a reaction volume of 10-200 μl in smallreaction tubes (0.2-0.5 ml volumes) in an apparatus referred to as athermal cycler. The thermal cycler heats and cools the reaction tubes toachieve the temperatures required at each of the following steps of thereaction:

Denaturation Step:

This step consists of heating the reaction to usually around 94-98° C.for approximately 20-30 seconds. It causes denaturation of the DNAtemplate by disrupting the hydrogen bonds between complementary bases,yielding single strands of DNA.

Annealing Step:

The reaction temperature is lowered to usually around 50-65° C. forapproximately 20-40 seconds allowing annealing of the primers to thesingle-stranded DNA template. Stable DNA-DNA hydrogen bonds are formedwhen the primer sequence closely matches the template sequence. Thepolymerase (e.g., Taq polymerase) binds to the primer-template hybridand begins DNA synthesis.

Extension Step:

The temperature at this step depends on the DNA polymerase used. Taqpolymerase has its optimum activity temperature at about 75° C., andcommonly a temperature of 72° C. is used with this enzyme. At this stepthe DNA polymerase synthesizes a new DNA strand complementary to the DNAtemplate strand by adding dNTPs that are complementary to the templatein the 5′ to 3′ direction, condensing the 5′-phosphate group of thedNTPs with the 3′-hydroxyl group at the end of the extending DNA strand.The extension time depends on the DNA polymerase used and on the lengthof the DNA fragment to be amplified. Under optimum conditions, at eachextension step the amount of the target DNA is doubled, leading toexponential amplification of the specific target DNA.

PCR usually includes of a series of 20 to 40 repeated cycles of theabove-described denaturation, annealing, and extension steps. Thecycling is often preceded by a single initialization step at a hightemperature (>90° C.), and followed by one final hold at the end forfinal product extension or brief storage. The initialization stepconsists of heating the reaction to a temperature of usually 94-96° C.(or 98° C. if extremely thermostable polymerases are used), which isheld for 1-9 minutes. The final hold usually occurs at 4-15° C. for anindefinite time and may be employed for short-term storage of thereaction. The temperatures used and the length of time they are appliedin each cycle depend on a variety of parameters. These include theenzyme used for DNA synthesis, the concentration of divalent ions anddNTPs in the reaction, and the melting temperature (T_(m)) of theprimers.

Following thermal cycling, agarose gel electrophoresis may be employedfor size separation of the PCR products to check whether PCR amplifiedthe target DNA fragment. The size(s) of the PCR products is determinedby comparison with a molecular weight marker, which contains DNAfragments of known size, run on the gel alongside the PCR products.

There are many applications of PCR. For example, real-time PCR (RT-PCR)is an established tool for DNA quantification that measures theaccumulation of DNA product after each round of PCR amplification. Thus,RT-PCR enables both detection and quantification of one or more specificsequences in a DNA sample (as absolute number of copies or relativeamount when normalized to DNA input or additional normalizing genes).Such quantitative PCR methods allow the estimation of the amount of agiven sequence present in a sample—a technique often applied toquantitatively determine levels of gene expression.

RT-PCR procedure follows the general principle of PCR. However, inRT-PCR, the amplified DNA is detected as the reaction progresses in realtime (whereas in standard PCR, the product of the reaction is detectedat the end of the reaction). One common method for detection of productsin RT-PCR is the use of nonspecific fluorescent dyes that intercalatewith double-stranded DNA (dsDNA). For example, SYBR Green is anasymmetrical cyanine dye that binds to dsDNA, and the resulting DNA-dyecomplex absorbs blue light (λ_(max)=488 nm) and emits green light(λ_(max)=522 nm). The DNA-binding dye, such as SYBR Green, binds to alldsDNA in PCR, causing fluorescence of the dye. An increase in DNAproduct during PCR therefore leads to an increase in fluorescenceintensity and is measured at each cycle, thus allowing DNAconcentrations to be quantified.

Another method for detection of products in RT-PCR is the use ofsequence-specific DNA probes, which are oligonucleotides that arelabeled with a fluorescent reporter that permits detection afterhybridization of the probe with its complementary DNA target. Many ofthese probes include a DNA-based probe having a fluorescent reporter(e.g., at one end of the probe) and a quencher of fluorescence (e.g., atthe opposite end of the probe). The close proximity of the reporter tothe quencher prevents detection of its fluorescence; breakdown of theprobe by the 5′ to 3′ exonuclease activity of the Taq polymerase breaksthe reporter-quencher proximity and thus allows unquenched emission offluorescence, which can be detected. An increase in the product targetedby the reporter probe at each PCR cycle therefore causes a proportionalincrease in fluorescence due to the breakdown of the probe and releaseof the reporter. Examples of such probes well known to those of ordinaryskill in the art are molecular beacon probes, TaqMan® probes, andScorpion™ probes.

Molecular beacons are single-stranded oligonucleotide probes that form ahairpin-shaped stem-loop structure. The loop contains a probe sequencethat is complementary to a target sequence in the PCR product. The stemis formed by the annealing of complementary sequences that are locatedon either side of the probe sequence. A fluorophore and quencher arecovalently linked to the ends of the hairpin. Upon hybridization to atarget sequence the fluorophore is separated from the quencher andfluorescence increases. Hybridization usually occurs after unfolding ofthe hairpin and product duplexes in the denaturation step of the nextPCR cycle.

TaqMan® probes are single-stranded unstructured oligonucleotides. Theyhave a fluorophore attached to the 5′ end and a quencher attached to the3′ end. When the probes are free in solution, or hybridized to a target,the proximity of the fluorophore and quencher molecules quenches thefluorescence. During PCR, when the polymerase replicates a template onwhich a TaqMan® probe is bound, the 5′-nuclease activity of thepolymerase cleaves the probe. Upon cleavage, the fluorophore is releasedand fluorescence increases.

Scorpion™ probes use a single oligonucleotide that consists of ahybridization probe (stem-loop structure similar to molecular beacons)and a primer linked together via a non-amplifiable monomer. The hairpinloop contains a specific sequence that is complementary to the extensionproduct of the primer. After extension of the primer during theextension step of a PCR cycle, the specific probe sequence is able tohybridize to its complement within the extended portion when thecomplementary strands are separated during the denaturation step of thesubsequent PCR cycle, and fluorescence will thus be increased (in thesame manner as molecular beacons).

However, there are drawbacks to the probes used to detect products inRT-PCR. For example, in the case of the use of nonspecific fluorescentdyes, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products,including nonspecific PCR products (such as “primer dimers”—i.e., primermolecules that have hybridized to each other). This interferes with andprevents accurate quantification of the intended target DNA sequence.

The use of sequence-specific DNA probes (e.g., molecular beacons,TaqMan®, and Scorpion™ probes) reduces or eliminates some of thedrawbacks inherent in nonspecific fluorescent dyes. For example,fluorescent reporter probes detect only the DNA containing the probesequence; therefore, use of the reporter probe significantly increasesspecificity, and enables quantification even in the presence ofnon-specific DNA amplification. Fluorescent probes can be used inmultiplex assays—for detection of several genes in the samereaction—based on specific probes with different-colored labels,provided that all targeted genes are amplified with similar efficiency.The specificity of fluorescent reporter probes also preventsinterference of measurements caused by primer dimers.

However, there are also drawbacks with these sequence-specific probes.For example, there are several disadvantages with molecular beacons.First, they require two bulky and costly tags (fluorophore andquencher). Second, the assay requires a separate probe for each template(i.e. mRNA), which dramatically increases the design effort and expense.Third, the mechanism uses separate binding sites for primer and probesequences, which introduces another component (probe oligonucleotide) toan already complex reaction, and adds additional design limitations dueto the need to avoid interactions between the probe and primers. Fourth,hybridization of the probe requires heating steps to unfold the productduplex and hairpin. Consequently, molecular beacons can't be used underisothermal conditions. Fifth, design of the probe requires considerableeffort and knowledge of nucleic acid thermodynamics. And sixth, probehybridization involves a bimolecular probe-primer system. This makes thereaction entropically unfavorable, slows down hybridization, andcomplicates product detection at exponential growth. The hybridizationis much faster and efficient with a monomolecular probe-primer system[as described in Whitcombe, D. et al. (1999) Detection of PCR productsusing self-probing amplicons and fluorescence. Nat Biotechnol, 17,804-807, incorporated by reference herein in its entirety].

All of the shortcomings listed above for molecular beacons hold true forTaqMan® probes. And, an additional disadvantage of TaqMan® probes isthat they require the 5′-nuclease activity of the DNA polymerase usedfor PCR.

Additionally, many of the shortcomings listed for molecular beacons holdtrue for Scorpion™ probes. First, they require two bulky and costly tags(fluorophore and quencher). Second, the assay requires a separate probefor each template (i.e. mRNA), which dramatically increases the designeffort and expense. Third, the mechanism uses separate binding sites forprimer and probe sequences, which introduces another component (probeoligonucleotide) to an already complex reaction, and adds additionaldesign limitations due to the need to avoid interactions between theprobe and primers. Fourth, hybridization of the probe requires heatingsteps to unfold the product duplex and hairpin. Consequently, molecularbeacons can't be used under isothermal conditions. And fifth, design ofthe probes requires considerable effort and knowledge of nucleic acidthermodynamics.

Further, fluorescent reporter probes do not prevent the inhibitoryeffect of the primer dimers, which may depress accumulation of thedesired products in the reaction.

Apart from any problems with probes listed above, there are furtherproblems inherent in PCR and RT-PCR. For example, one of the mostimportant factors limiting the yield of specific product is thecompetition between primer binding and self-annealing of the product. Atthe initial stage of PCR, product molecules are at low enoughconcentrations that product self-annealing does not compete with primerbinding and amplification proceeds at an exponential rate. However, withaccumulation of product DNA, self-annealing becomes dominant and PCRslows down and eventually DNA amplification ceases.

Temperature cycling is another limitation of PCR since it requiresexpensive instrumentation for thermocycling and complicates rapiddetection of pathogens in the field and at point-of-care. In addition,rapid temperature changes facilitate product mis-priming and affectstability of the polymerases.

To decrease certain of the above-described drawbacks, such as the costof probe synthesis, several attempts have been made to use intrinsicfluorescence of nucleotides. For example, 2-aminopurine (2Ap) has beenused as a label for the detection of product in RT-PCR. 2Ap is afluorescent analog of adenosine and has been used as a site-specificprobe of nucleic acid structure and dynamics because it base pairs withcytosine in a wobble configuration or with thymine in a Watson-Crickgeometry. 2Ap has been incorporated into stem-loop probes [as describedin Walker, G. T. et al. (1992) Strand displacement amplification—anisothermal, in vitro DNA amplification technique. Nucleic acidsresearch, 20, 1691-1696]. Upon hybridization to a target sequence, aseveral-fold increase in fluorescence was observed due to the change of2Ap from a double-helix (quenched state) to a single-stranded region(emitted state). However, the sensitivity of the 2Ap probes isinsufficient for accurate monitoring of PCR, since 2Ap is stillsignificantly quenched in single strands.

SUMMARY OF THE INVENTION

Certain exemplary aspects of the invention are set forth below. Itshould be understood that these aspects are presented merely to providethe reader with a brief summary of certain forms the invention mighttake and that these aspects are not intended to limit the scope of theinvention. Indeed, the invention may encompass a variety of aspects thatmay not be explicitly set forth below.

In an overarching aspect, the present invention provides a newamplification system that reduces or eliminates the shortcomings of PCRand RT-PCR described above. For example, as described above, PCR islimited by competition between primer binding and undesiredself-annealing of target DNA. One aspect of the present inventioninhibits self-annealing by providing at least one primer, such as anoligonucleotide primer, for amplification of a target nucleic acid(e.g., DNA), wherein the primer is adapted to conform into a structurethat can dissociate from a DNA duplex structure in the absence ofheating. In other words, the primer includes a sequence that naturallyconforms into a structure, such as a quadruplex structure (or any othernon-B DNA configuration or other DNA structure) in which intramolecularbase pairing allows or causes the primer to dissociate from the doublestranded DNA—of which it is one strand—and form its particularstructure. This structure may be referred to herein as a “dissociativestructure” or “dissociative conformation” or the like. This example ofsuch a primer may conform into the structure such as during theextension step of PCR. As this occurs, the primer necessarily separatesfrom its binding site on the target DNA sequence while the extendingportion (DNA polymerase adding dNTPs to the sequence) remains bound (atleast temporarily) in a double-stranded configuration. In other words,one aspect of the invention is that the primer can be of any sequencewherein its free energy can drive PCR upon extension of the primer orupon interaction with a polymerase during PCR.

Further, the primers used in this aspect of the present invention may beuniversal. In other words, each primer in the primers used in thereaction includes the same sequence (or a substantially similar sequencethat allows amplification to occur). As is known to those of ordinaryskill in the art, in standard PCR at least two different primers (i.e.,having two different sequences) are used (i.e., a first set of primers,wherein each primer of the first set includes the same or similarsequence, and a second set of primers wherein each primer of the secondset includes the same or similar sequence, that sequence being differentfrom the sequence of the primers in the first set). The need for the twosets of primers is to provide for amplification using each of the singlestrands from the DNA. Thus, one strand will be replicated using primersfrom the first set. The second strand (which is complementary to thefirst strand) will also be replicated. However, as that second strand iscomplementary to the first strand, the primers of the second set may becomplementary to the primers of the first set. This creates the problemof primer-dimers (when the primers hybridize to one another—rather thanto the target template denatured DNA strands). However, in the presentinvention, each of the primers used have the same or similar sequence.There is no second set of complementary primers that is needed (as willbe described in greater detail below). As such, there are no othercomplementary primers for the primers of the present application tohybridize to, thus eliminating the problem of primer dimers. Further, asthe end being extended is currently bound with the target region,self-annealing of product is eliminated. And, the original primerbinding site on the target DNA region is open for binding of anotherprimer.

While “at least one primer” and a site being “open for binding ofanother primer” are discussed herein, it will be recognized by those ofordinary skill in the art that the “at least one primer” and the“another primer” may have the same sequence (or substantially similarsequence) as it is known that PCR employs multiple copies of a primerfor amplification of a target DNA sequence. Further, as is known tothose of ordinary skill in the art, the reaction components generallyinclude multiple copies of primers. Thus, it will be understood that “atleast one primer” or “one primer” or “a primer” or “the primer” or likereferences may refer to a single primer, or a primer among a set ofmultiple copies of the same or similar primers. Further, while variousPCR procedures described herein are discussed as amplifying DNA, thoseof ordinary skill in the art will recognize that does not limit thedisclosure to those seeking DNA sequences, as procedures such as reversetranscription PCR are well known, wherein reverse transcriptase reversetranscribes RNA into cDNA, which is then amplified by PCR.

In one aspect, the primer(s) may be based on any sequence that iscapable of forming a quadruplex structure (or other structure thatallows or causes the primer to dissociate from a double-stranded DNA andform the particular structure—e.g., a quadruplex—such as during anextension step of PCR). As is known to those of ordinary skill in theart, due to their base pairing properties, nucleic acid sequences canoften form specific structures under certain solution conditions. Forexample, in the presence of certain metal ions (e.g., K⁺), short guanine(G)-rich sequences fold into a structure known as a G-quartet orquadruplex. Quadruplexes are very stable and biophysical studies haveshown that they possess intrinsic optical properties (e.g., absorb lightat 300 nm) that distinguish them from other secondary structures.Previously, quadruplex-formation assays have been developed that exploitthis unique quadruplex signature to study enzymes that cleave DNA[Kankia, B. I. (2006) A real-time assay for monitoring nucleic acidcleavage by quadruplex formation, Nucleic acids research, 34, p. 141] orfacilitate strand-exchange reactions [Kankia, B. I. (2004) Opticalabsorption assay for strand-exchange reactions in unlabeled nucleicacids, Nucleic acids research, 32, p. 154]. Briefly, when G-richsequences with the potential to form a quadruplex are incorporated intoDNA substrates they are initially in the quenched state. Upon enzymaticactivity (i.e., strand cleavage or strand-exchange) the releasedsequence folds into a quadruplex and becomes visible when monitored byabsorption or fluorescence spectroscopy.

One aspect of the present invention, then, uses the free energy of DNAquadruplexes (or other non-quadruplex conformations) to driveunfavorable (endergonic) reactions of nucleic acids (e.g., isothermalPCR). The key point of such reactions is that some sequences—e.g., someG-rich sequences—are capable of forming quadruplexes (or otherconformations) with significantly more favorable thermodynamics than thecorresponding DNA duplexes. The sequences are incorporated within DNAduplexes, which after interaction with an initiator (e.g., DNApolymerase) self-dissociate from the complementary strand and fold intoquadruplexes (or other conformations). The energy of formation of thenon B-DNA structure, or other DNA structure is used to drive PCR atsubstantially constant temperature.

Another aspect of the present invention then provides a primer orprimers having sequences that are capable of forming structures todissociate from a DNA duplex, such as a quadruplex. Thus, in certainembodiments, the primer may include a sequence that is generally basedon a sequence in the form of d(G₃₊N₁₋₇G₃₊N₁₋₇G₃₊N₁₋₇G₃). In certainembodiments, the sequence may be a G-rich sequence. And, in oneparticular embodiment, the primer(s) may be based on the sequenceGGGTGGGTGGGTGGGT [SEQ. ID. NO. 1] [“(GGGT)₄” ], which is capable offorming a very stable quadruplex, even in the presence of acomplementary strand. By being “based on” the sequence (GGGT)₄ [SEQ. ID.NO. 1], those of ordinary skill in the art will recognize thatsubstitutions and/or deletions may be made to this base sequence, solong as the resulting primer based on the (GGGT)₄ [SEQ. ID. NO. 1]sequence remains able to conform into a quadruplex structure, either onits own or during an extension step of a PCR process. The particularprimers may be shorter sequences based on the (GGGT)₄ [SEQ. ID. NO. 1]sequence. These primers thus may be unable to fold into a quadruplex ontheir own, but will fold into a quadruplex structure after polymeraseelongation and dissociation from the target site, and will stay foldedat annealing of the next cycle. This process is referred to herein as“quadruplex priming amplification” (QPA). Because QPA inhibits productself-annealing and increases the number of PCR cycles within theexponential growth phase, this aspect of the present invention improvesefficiency of PCR by elongating the window of exponential amplification.While a particular sequence capable of forming such a quadruplexstructure—e.g., (GGGT)₄ [SEQ. ID. NO. 1]—is described in this embodiment(and while this process is referred to as “Quadruplex PrimingAmplification” or “QPA”), it will be recognized by those of ordinaryskill in the art that the invention is not limited to the particularsequence, or to a sequence that forms a quadruplex, and that anysequence that dissociates from a DNA duplex to form another structure(whether quadruplex or a structure other than a quadruplex) may be usedin accordance with the principles of the present invention.

Further, since the exemplary quadruplex structure is more stable thanits corresponding duplex, unfolding of the duplex or release of targetfor the coming primers can occur without the need of substantialtemperature change or any temperature change. In other words, instandard PCR, following the extension step, the DNA is in a duplex form.The next cycle then begins by raising the temperature to a point thatthe double-stranded DNA again denatures (i.e., separates into singlestrands). This is necessary in order to provide the separated sense andanti-sense DNA strands for primer binding (to each of the strands),followed by elongation during the next extension step (once thetemperature of the reaction is reduced). However, by using primers basedon the (GGGT)₄ [SEQ. ID. NO. 1] sequence, the primers and extendingnucleotides that are added during the extension step naturally conforminto the quadruplex structure. As this occurs, the primer (forming thequadruplex structure) naturally separates from the target DNA sequencecomplementary to the primer, thereby leaving the target regioncomplementary to the primer exposed in single-stranded form for bindingof the next primer. This occurs without requiring raising of thetemperature to denature the strands from one another. Thus, QPA canproceed under isothermal conditions. And so, the isothermal DNAamplification provided by the present invention does not requireexpensive instrumentation for thermocycling and allows DNA amplificationin the field and at point-of-care.

Thus, another aspect of the present invention provides an isothermicprocess for amplifying at least one target nucleic acid sequencecontained in a nucleic acid or a mixture of nucleic acids. The processincludes treating a nucleic acid or a mixture of nucleic acids with atleast one oligonucleotide primer adapted to conform into a quadruplexstructure during an extension step of a polymerase chain reaction, underisothermic conditions. During this isothermic process, for the at leastone nucleic acid sequence being amplified, an extension product of theat least one oligonucleotide primer is synthesized which iscomplementary to a strand from the nucleic acid or mixture of nucleicacids. In this process, the at least one oligonucleotide primer isselected so as to be sufficiently complementary to the strand from thenucleic acid or a mixture of nucleic acids to hybridize therewith suchthat the extension product synthesized from the at least oneoligonucleotide primer, when it is separated from its complement, canserve as a template for further synthesis of an extension product ofanother oligonucleotide primer.

Further, as described above, a drawback of current RT-PCR-specificquantification systems is that they use FRET-based applications (FörsterResonance Energy Transfer), which require costly synthesis andconsiderable effort to design a sensitive probe. A quantificationmechanism that uses intrinsic fluorescence of primers will significantlysimplify the detection process. Thus, another aspect of the presentinvention includes a quadruplex forming primer having a sequence withincorporated 2Ap, which emits strong fluorescence upon quadruplexformation. In one particular embodiment, the 2Ap can be incorporatedinto a primer based on the (GGGT)₄ [SEQ. ID. NO. 1] sequence as follows:GGG(2Ap)GGGTGGGTGGG (referred to herein as “2Ap-G3T”) [SEQ. ID. NO. 10].Thus, another aspect of the present invention provides a QPA-basedreal-time quantification system (RT-QPA) using 2Ap as a sensitive probefor quantification.

Thus, this aspect of the present invention provides a real-timequantification PCR method for detecting amplification of a targetnucleic acid. The method includes treating a nucleic acid or a mixtureof nucleic acids with at least one oligonucleotide primer adapted toconform into a quadruplex structure (or other dissociative structure)during an extension step of a polymerase chain reaction, underconditions such that for the at least one nucleic acid sequence beingamplified an extension product of the at least one oligonucleotideprimer is synthesized which is complementary to a strand from thenucleic acid or a mixture of nucleic acids. In the method, the at leastone oligonucleotide primer includes a label that is quenched when the atleast one oligonucleotide primer is in a non-quadruplex conformation andthat is detectable when the at least one oligonucleotide primer is in aquadruplex conformation.

Thus, for example, after polymerase elongation in RT-QPA, specificallydesigned guanine-rich primer(s) are capable of forming quadruplexes withsignificantly more favorable thermodynamics than the corresponding DNAduplexes. As a result, target sequences are always accessible for theprimers since their complementary strands are trapped in a quadruplexconformation and DNA amplification can proceed under isothermalconditions. In addition, 2Ap nucleotides incorporated and fully quenchedwithin the primers regain maximum emission upon quadruplex formationallowing very simple and accurate detection of product DNA. Thus, thequadruplex priming amplification (QPA) (i) lacks a productself-annealing; (ii) can proceed under isothermal conditions; and (iii)uses intrinsic fluorescence of primers for quantification of DNAproducts.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the general description of the invention given above andthe detailed description of the embodiments given below, serve toexplain the principles of the present invention.

FIG. 1 is a graph showing a typical RT-PCR curve.

FIG. 2 is a schematic of structures and modes of action of previousprobes with panel A showing a molecular beacon, panel B showing aTaqMan®, and panel C showing a Scorpion™ probe.

FIG. 3 is a schematic of a DNA G-quartet.

FIG. 4 is a schematic illustration of the QPA process.

FIG. 5 shows the incorporation of the QPA target site (dotted lines) intemplates by attachment of quadruplex forming sequences (dashed lines)to primers.

FIG. 6 is a CD spectra of (GGGT)₄ (black line), GGGTGGGTGGGTGGG[“(GGGT)₃GGG” ] (-□-) [SEQ. ID. NO. 2], and 2Ap-G3T (-∘) [SEQ. ID. NO.10] using 5 μM concentration in 50 mM KCl, 2 mM MgCl₂, 20 mM Tris-HCl,pH 8.7 at 20° C.

FIG. 7 is fluorescence spectra at 20° C. of 2Ap-G3T [SEQ. ID. NO. 10] in50 mM KCl in the absence of complement (-∘-) and in the presence of thecomplementary strand in 50 mM CsCl (black line), showing that under thelatter conditions, 2Ap-G3T forms a perfect duplex with completelyquenched 2Ap.

FIG. 8 shows fluorescence melting curves of single-strandedoligonucleotides, where G3T-ss15 is 2Ap-G3T [SEQ. ID. NO. 10], G3T-ss14is GGG(2Ap)GGGTGGGTGG [SEQ. ID. NO. 11], and G3T-ss13 isGGG(2Ap)GGGTGGGTG [SEQ. ID. NO. 12] in 50 mM monocations.

FIG. 9 shows fluorescence melting curves of G3T-ds15 duplex (i.e.,GGG(2Ap)GGTGGGTGGG (“2Ap-G3T”) [SEQ. ID. NO. 10] in duplex form with itscomplementary strand CCCACCCACCCTCCC [SEQ. ID. NO. 3]), wherein theblack and squared lines correspond to heating and cooling (at 1° C./minrate), respectively.

FIG. 10 shows UV melting curves of G3T-ds15 [SEQ. ID. NO. 10] andG3T-ds13 (i.e., GGG(2Ap)GGGTGGGTG [SEQ. ID. NO. 12] in duplex form witha complementary strand CCCTCCCACCCACCC [SEQ. ID. NO. 3]) duplexes in thepresence (-∘- and -□-) and absence (-Δ- and black line) of K⁺ ions.

FIG. 11 shows fluorescence melting curves of a 2Ap-G3T [SEQ. ID. NO. 10]duplex in 15 mM KCl, 35 mM CsCl, 2 mM MgCl₂, 20 mM Tris-HCl, pH 8.7wherein the black and squared lines correspond to heating and cooling(at 1° C./min rate), respectively.

FIG. 12 shows fluorescence melting curves of 2Ap-G3T [SEQ. ID. NO. 10](black line) and its truncated versions [i.e., G3T-ss14 [SEQ. ID. NO.11] (-∘-) and G3T-ss13[SEQ. ID. NO. 12] (-Δ-)] in 50 mM KCl.

FIG. 13 shows isothermal QPA at 55° C. at 50 nM (black line) and 5 nM(-∘-) template, 1 μM primer, 200 μM dNTPs and 5U Taq in 100 μl bufferindicated in FIG. 11.

FIG. 14 includes schematics showing the sequence of QPA primer andtemplates used in the reactions (panel A), exponential QPA (in panel B),and linear QPA (in panel C).

FIG. 15A shows isothermal QPA using 100 nM template, 1 μM primer, 200 μMdNTPs and 5U Taq in 100 μL buffer (25 mM KCl, 25 mM CsCl, 2 mM MgCl₂).

FIG. 15B is a photograph of agarose gel electrophoresis of the QPAproducts shown in FIG. 15A visualized by ethidium bromide, where lanes 2and 3 are product at 75° C., lanes 3 and 4 are product at 72° C., lanes5 and 6 are product at 70° C. after 10 hours of kinetics, lanes 7 and 8are product at 70° C. after 70 min of kinetics, and lanes 9 and 10 are 1μM positive control (a chemically synthesized product duplex).

FIG. 16A is a graph showing thermo-cycling QPA of exponential (circles)and linear (squares) templates at 70° C. for 3 min and 94° C. for 1 min,where the reaction mixtures (100 μl) include 1 μM primer, 200 μM dNTPs,5U Taq, 5 mM KCl, 45 mM CsCl, 2 mM MgCl₂, 10 mM Tris-HCl at 10 fMtemplate.

FIG. 16B is a graph showing thermo-cycling QPA of exponential (circles)and linear (squares) templates at 70° C. for 3 min and 94° C. for 1 min,where the reaction mixtures (100 μl) include 1 μM primer, 200 μM dNTPs,5U Taq, 5 mM KCl, 45 mM CsCl, 2 mM MgCl₂, 10 mM Tris-HCl at 1 nMtemplate.

FIG. 17A is a graph showing isothermal QPA at 72° C. using 10 pMtemplate, 1 μM primer, 200 μM dNTPs and 5U Taq in 100 μL buffer (25 mMKCl, 25 mM CsCl, 2 mM MgCl₂).

FIG. 17B is a photograph of agarose gel electrophoresis of QPA productsshown in FIG. 17A visualized by ethidium bromide, where lane 1 ischemically synthesized product duplex, lane 2 is product at 30 min, lane3 is product at 80 min, lane 4 is product at 200 min, and lane 5 isproduct at 700 min.

FIG. 18 shows thermocycling QPA after attachment of QPA-target sites toa pUC18 cloning region (101-nt), and where the negative control(squares) does not include initial PCR primers.

FIG. 19 shows isothermal RT-QPA at 72° C. using the shown template andtarget with a reaction mixture (100 μl): 1 μM primer, 20 μM dNTPs, 5UTaq, 50 mM KCl, 2 mM MgCl₂, 20 mM Tris-HCl, pH 8.7 at 50 fM (-Δ-), 1 pM(-∘-), 5 pM (-□-), and 10 pM (black line) template DNA.

FIG. 20 includes schematic diagrams showing two possible structures of(GGGT)₄ [SEQ. ID. NO. 10] with panel A showing an anti-parallelconformation based on NMR work, and with panel B showing a parallelconformation suggested on the bases of thermodynamic and spectralstudies.

FIG. 21 is a graph of fluorescence spectra of GGG(6MI)GGGCGGGCGGG [SEQ.ID. NO. 21] without and with its complementary strand.

DETAILED DESCRIPTION

One or more specific embodiments of the present invention will bedescribed below. In an effort to provide a concise description of theseembodiments, all features of an actual implementation may not bedescribed in the specification. It should be appreciated that in thedevelopment of any such actual implementation, as in any engineering ordesign project, numerous implementation-specific decisions must be madeto achieve the developers' specific goals, such as compliance withsystem-related and business-related constraints, which may vary from oneimplementation to another. Moreover, it should be appreciated that sucha development effort might be complex and time consuming, but wouldnevertheless be a routine undertaking of design, fabrication, andmanufacture for those of ordinary skill having the benefit of thisdisclosure.

As described above, in an overarching aspect, the present inventionprovides a new amplification system that reduces or eliminates theshortcomings of PCR described above. For example, as described above,PCR is limited by competition between primer binding and undesiredself-annealing of target DNA. Referring to FIG. 1, which shows a typicalPCR, at the initial stage of PCR, product molecules are at low enoughconcentrations that product self-annealing does not compete with primerbinding and amplification proceeds at an exponential rate (see the ACsegment, FIG. 1; the AB segment corresponds to exponential phaseundetectable by fluorescence measurements). However, with accumulationof product DNA, self-annealing becomes dominant and PCR slows (CDsegment, FIG. 1) and eventually DNA amplification ceases (plateau, FIG.1).

One aspect of the present invention inhibits this self-annealing byproviding at least one primer, such as an oligonucleotide primer, foramplification of a target nucleic acid (e.g., DNA), wherein the primeris adapted to conform into a structure that can dissociate from a DNAduplex structure in the absence of heating. In other words, the primerincludes a sequence that naturally conforms into a structure, such as aquadruplex structure (or any other non-B DNA configuration or other DNAstructure) in which intramolecular base pairing allows or causes theprimer to dissociate from the double stranded DNA—of which it is onestrand—and form its particular structure. This structure may be referredto herein as a “dissociative structure” or “dissociative conformation”or the like. As one non-limiting example, the primer may be adapted toconform into a quadruplex structure. The primer may conform into thequadruplex structure during an extension step of PCR. As this occurs,the primer necessarily separates form its binding site on the target DNAsequence while the extending portion (DNA polymerase adding dNTPs to thesequence) remains bound (at least temporarily) in a double-strandedconfiguration. Again, it will be recognized by those of skill in the artthat quadruplex structures are merely exemplary, and the primer may formany other structure that can disassociate from any DNA configuration ofwhich it is a part.

Further, the primers used in this aspect of the present invention may beuniversal. In other words, each primer in the primers used in thereaction includes the same sequence (or a substantially similar sequencethat allows amplification to occur). As is known to those of ordinaryskill in the art, in standard PCR at least two different primers (i.e.,having two different sequences) are used (i.e., a first set of primers,wherein each primer of the first set includes the same or similarsequence, and a second set of primers wherein each primer of the secondset includes the same or similar sequence, that sequence being differentfrom the sequence of the primers in the first set). The need for the twosets of primers is to provide for amplification using each of the singlestrands from the DNA. Thus, one strand will be replicated using primersfrom the first set. The second strand (which is complementary to thefirst strand) will also be replicated. However, as that second strand iscomplementary to the first strand, the primers of the second set may becomplementary to the primers of the first set. This creates the problemof primer-dimers (when the primers hybridize to one another—rather thanto the target template denatured DNA strands). However, in the presentinvention, each of the primers used have the same or similar sequence.There is no second set of complementary primers that is needed. As such,there are no other complementary primers for the primers of the presentapplication to hybridize to, thus eliminating the problem of primerdimers. Further, as the end being extended is currently bound with thetarget region, self-annealing of product is eliminated. And, theoriginal primer binding site on the target DNA region is open forbinding of another primer.

While “at least one primer” and a site being “open for binding ofanother primer” are discussed herein, it will be recognized by those ofordinary skill in the art that the “at least one primer” and the“another primer” may have the same sequence (or substantially similarsequence) as it is known that PCR employs multiple copies of a primerfor amplification of a target DNA sequence. Further, as is known tothose of ordinary skill in the art, the reaction components generallyinclude multiple copies of primers. Thus, it will be understood that “atleast one primer” or “one primer” or “a primer” or “the primer” or likereferences may refer to a single primer, or a primer among a set ofmultiple copies of the same or similar primers. Further, while variousPCR procedures described herein are discussed as amplifying DNA, thoseof ordinary skill in the art will recognize that does not limit thedisclosure to those seeking DNA sequences, as procedures such as reversetranscription PCR are well known, wherein reverse transcriptase reversetranscribes RNA into cDNA, which is then amplified by PCR.

As described above, in one aspect, the primer or primers may be based onany sequence that is capable of forming a structure that allows orcauses the primer to dissociate from a duplex form, such as underisothermic conditions. And, as described above, one such structure whichallows such dissociation is a quadruplex structure. As previouslydiscussed, such quadruplex structures are commonly formed by sequencesrich in guanine residues. Thus, much of the discussion below is directedto G-rich sequences for primers, which are capable of forming suchquadruplex structures. However, it will be appreciated by those ofordinary skill in the art that the general and particular sequencesdescribed below, and the particular structures, such as quadruplexstructures, described below, are merely exemplary and that there may beother useful sequences that form structures which allow dissociationfrom a double-stranded DNA form in accordance with the principles of thepresent invention.

As is known to those of ordinary skill in the art, quadruplexes arehigh-ordered nucleic acid structures (DNA or RNA) formed from G-richsequences that are built around tetrads of hydrogen bonded guaninebases. Thus, in order to provide primers that can form quadruplexes, theprimers of this aspect of the present invention may be designed with asequence having a G content of a high enough amount (or to obtain a highenough amount) to allow the primer to conform into a quadruplexstructure, such as during an extension step of PCR. In one embodiment,the G content of the sequence of a primer of this aspect of the presentinvention is equal to or greater than 70%. In another embodiment, the Gcontent may be equal to or greater than 75%. More specifically, in oneembodiment, the primer may be an oligonucleotide having a sequence basedon GGGTGGGTGGGTGGGT [SEQ. ID. NO. 1] [“(GGGT)₄” ]. This sequence canform into a quadruplex. However, it will be recognized by those ofordinary skill in the art that primers for use in this aspect of thepresent invention do not have to include the exact (GGGT)₄ [SEQ. ID. NO.1] sequence. By being “based on” the sequence (GGGT)₄ [SEQ. ID. NO. 1],those of ordinary skill in the art will recognize that substitutionsand/or deletions may be made to this base sequence, so long as theresulting primer based on the (GGGT)₄ [SEQ. ID. NO. 1] sequence remainsable to conform into a quadruplex structure, either on its own or duringan extension step of a PCR process. For example, as described above,other sequences may form quadruplexes provided they include a guanineamount that is sufficient to form such quadruplexes. Further, thesequences do not have to be based on (GGGT)₄ [SEQ. ID. NO. 1], as thereare other formulas that the sequences may be based on. One example ofsuch a formula is d(G₃₊N₁₋₇G₃₊N₁₋₇G₃₊N₁₋₇G₃).

In a particular embodiment, the at least one oligonucleotide primer isbased on the sequence GGGTGGGTGGGTGGGT [SEQ. ID. NO. 1] [“(GGGT)₄” ],which is capable of forming a very stable quadruplex, even in thepresence of the complementary strand. The particular primers may beshorter versions of the (GGGT)₄ [SEQ. ID. NO. 1] sequence. These primersare thus unable to fold into a quadruplex on their own, but will foldinto a quadruplex structure after polymerase elongation and dissociationfrom the target site, and will stay folded at annealing of the nextcycle. Because QPA inhibits product self-annealing and increases thenumber of PCR cycles within the exponential growth phase, this aspect ofthe present invention improves efficiency of PCR by elongating thewindow of exponential amplification.

As described above, one aspect of the present invention includes the useof novel primers that form quadruplexes upon elongation. Quadruplexesare high-ordered DNA and RNA structures formed from G-rich sequencesthat are built around tetrads of hydrogen bonded guanine bases. Thesynthetic polynucleotides poly(dG) and poly(G) were determined to formfour-stranded helical structures, with the G-tetrads stacked on oneanother, analogous to Watson-Crick base pairs in duplex DNA.

Quadruplexes can be formed from one, two, or four separate strands ofDNA (or RNA) and can display a wide variety of topologies, which are inpart a consequence of the various possible combinations of stranddirections, as well as variations in loop size and sequence. They can bedefined in general terms as structures formed by a core of at least twostacked G-tetrads, which are held together by loops arising from theintervening mixed sequence nucleotides that are not usually involved inthe tetrads themselves. The combination of the number of stackedG-tetrads, the polarity of the strands, and the location and length ofthe loops would be expected to lead to a plurality of G quadruplexstructures, as is found experimentally [as described in Burge, S. etal., Survey and Summary: Quadruplex DNA: sequence, topology, andstructure, Nucleic Acids Research, 2006, Vol. 34, No. 19, pp.5482-5415].

Quadruplex structures can be classified according to their strandpolarities and the location of the loops that link the guanine strandsfor quadruplexes formed either from a single strand or from two strands.Adjacent linked parallel strands require a connecting loop to link thebottom G tetrad with the top G tetrad leading to what is referred to as“propeller”-type loops (these are also sometimes termed“strand-reversal” loops). This feature has been found both in crystalstructures and in solution for quadruplexes formed from human telomericDNA sequences and in a number of nontelomeric quadruplexes [as describedin Burge, S. et al., Survey and Summary: Quadruplex DNA: sequence,topology, and structure, Nucleic Acids Research, 2006, Vol. 34, No. 19,pp. 5482-5415].

Quadruplexes are designated as “antiparallel” when at least one of thefour strands is antiparallel to the others. This type of topology isfound in the majority of bimolecular and many unimolecular quadruplexstructures determined to date. Two further types of loops have beenobserved in these structures, in addition to parallel loops. “Lateral”(sometimes termed “edgewise”) loops join adjacent G-strands. Two ofthese loops can be located either on the same or opposite faces ofquadruplex, corresponding to head-to-head or head-to-tail, respectively,when in bimolecular quadruplexes. The second type of antiparallel loop,the “diagonal” loop joins opposite G-strands. In this instance, thedirectionalities of adjacent strands must alternate between parallel andantiparallel, and are arranged around a core of four stacked G-tetrads[as described in Burge, S. et al., Survey and Summary: Quadruplex DNA:sequence, topology, and structure, Nucleic Acids Research, 2006, Vol.34, No. 19, pp. 5482-5415].

Further, in order to be detectable, such as for use in RT-PCR, theprimer may have a label incorporated therein. Such a label may be chosenfrom labels that are known to those of ordinary skill in the art. Suchlabels include, but are not limited to, fluorescent labels. In oneparticular embodiment, the primer may have a fluorescent labelincorporated therein. And in a particular embodiment, such a label mayinclude 2Ap.

The inclusion of a label in the novel primer of the present inventionovercomes many of the previously described drawbacks of current systems.As described above, RT-PCR quantification methods are based on the factthat the amount of target DNA produced and detected is directlyproportional to the initial amount of sample DNA during the exponentialgrowth phase. Since the fluorescence signal during the initial cycles istoo weak to be distinguished from the background fluorescence (see theAB segment of FIG. 1) only a narrow window of the exponential growthphase is used for quantification (see the BC segment of FIG. 1). Thus,the efficiency of RT-PCR would be improved by reducing the backgroundfluorescence (i.e., by the use of well-quenched probes beforedetection), and by a strong and immediate increase of fluorescence uponamplification, as well as by a longer exponential phase [as described inEdwards, K. J. et al. (2009) In Logan, J. et al. (eds.), Real-time PCR.Caister Academic Press, Norfolk, UK, pp. 85-93; Pfaffl, M. W. et al.(2009) In Logan, J. et al. (eds.), Real-time PCR. Caister AcademicPress, Norfolk, UK, pp. 65-83].

As described above currently, four main probes are used to monitorreal-time PCR [Lee, M. A., et al. (2009) In Logan, J. et al. (eds.),Real-time PCR. Caister Academic Press, Norfolk, UK, pp. 23-45]: (1) SYBRGreen [Becker, A. et al. (1996) A quantitative method of determininginitial amounts of DNA by polymerase chain reaction cycle titrationusing digital imaging and a novel DNA stain. Anal Biochem, 237,204-207], (2) molecular beacons [Tyagi, S. et al. (1998) Multicolormolecular beacons for allele discrimination. Nature biotechnology, 16,49-53; Tyagi, S. et al. (1996) Molecular beacons: probes that fluoresceupon hybridization. Nature biotechnology, 14, 303-308], (3) TaqMan®probes [Holland, P. M. et al. (1991) Detection of specific polymerasechain reaction product by utilizing the 5′-3′ exonuclease activity ofThermus aquaticus DNA polymerase. Proc Natl Acad Sci USA, 88,7276-7280], and (4) Scorpion™ probes [Whitcombe, D. et al. (1999)Detection of PCR products using self-probing amplicons and fluorescence.Nat Biotechnol, 17, 804-807]. And, as described previously, there aredrawbacks to each of these. SYBR Green is a dye that intercalates intodouble-stranded DNA nonspecifically resulting in fluorescence. AlthoughSYBR Green is inexpensive, sensitive and easy to use, it also binds toany double-stranded DNA including non-specific products or primerdimers.

Referring to FIG. 2, panel A, molecular beacons are single-strandedoligonucleotide probes that form a hairpin-shaped stem-loop structure.The loop contains a probe sequence (dashed line segment, FIG. 2, panelA) that is complementary to a target sequence in the PCR product. Thestem is formed by the annealing of complementary sequences that arelocated on either side of the probe sequence. A fluorophore (dottedcircle) and quencher (lined circle) are covalently linked to the ends ofthe hairpin. Upon hybridization to a target sequence the fluorophore isseparated from the quencher and fluorescence increases. Hybridizationusually occurs after unfolding of the hairpin and product duplexes inthe denaturation step of the next PCR cycle.

As described above, there are several disadvantages with molecularbeacons. First, they require two bulky and costly tags (fluorophore andquencher). Second, the assay requires a separate probe for each template(i.e. mRNA), which dramatically increases the design effort and expense.Third, the mechanism uses separate binding sites for primer and probesequences. This introduces another component (probe oligonucleotide) toan already complex reaction, and adds additional design limitations dueto the need to avoid interactions between the probe and primers. Fourth,hybridization of the probe requires heating steps to unfold the productduplex and hairpin. Consequently, molecular beacons can't be used underisothermal conditions. Fifth, design of the probes requires considerableeffort and knowledge of nucleic acid thermodynamics. And sixth, probehybridization involves a bimolecular probe-primer system. This makes thereaction entropically unfavorable, slows down hybridization andcomplicates product detection at exponential growth. The hybridizationis much faster and efficient with monomolecular probe-primer system [asdescribed in Whitcombe, D. et al. (1999) Detection of PCR products usingself-probing amplicons and fluorescence. Nat Biotechnol, 17, 804-807].

Referring now to FIG. 2, panel B, TaqMan® probes are single-strandedunstructured oligonucleotides designed to be complementary to a PCRproduct. They have a fluorophore attached to the 5′ end and a quenchercoupled to the 3′ end. When the probes are free in solution, orhybridized to a target the proximity of the fluorophore and quenchermolecules quenches the fluorescence. During PCR, when the polymerasereplicates a template on which a TaqMan® probe is bound, the 5′-nucleaseactivity of the polymerase cleaves the probe. Upon cleavage, thefluorophore is released and fluorescence increases. The shortcomingslisted above for molecular beacons hold true for TaqMan®. An additionaldisadvantage of TaqMan® probes is that they require the 5′-nucleaseactivity of the DNA polymerase used for PCR.

Referring now FIG. 2, panel C, Scorpion™ probes use a singleoligonucleotide that consists of a hybridization probe (stem-loopstructure similar to molecular beacons) and a primer (10) linkedtogether via a non-amplifiable monomer (12). The hairpin loop contains aspecific sequence that is complementary to the extension product of theprimer (dashed line). After extension of the primer during the extensionstep of a PCR cycle, the specific probe sequence is able to hybridize toits complement within the extended portion when the complementarystrands are separated during the denaturation step of the subsequent PCRcycle, and fluorescence will thus be increased (in the same manner asmolecular beacons). Many of the shortcomings listed for molecularbeacons hold true for Scorpion™ probes. First, they require two bulkyand costly tags (fluorophore and quencher). Second, the assay requires aseparate probe for each template (i.e. mRNA), which dramaticallyincreases the design effort and expense. Third, the mechanism usesseparate binding sites for primer and probe sequences. This introducesanother component (probe oligonucleotide) to an already complexreaction, and adds additional design limitations due to the need toavoid interactions between the probe and primers. Fourth, hybridizationof the probe requires heating steps to unfold the product duplex andhairpin. Consequently, molecular beacons can't be used under isothermalconditions. And fifth, design of the probes requires considerable effortand knowledge of nucleic acid thermodynamics.

In one aspect, the primer(s) may be based on any sequence that iscapable of forming a quadruplex structure (or other non-B DNAconformation or other DNA structure in which intramolecular base-pairingallows or causes the primer to dissociate from a double-stranded DNA andform the particular structure such as during an extension step of PCR).Non-B DNA conformations include triplexes. As is known to those ofordinary skill in the art, due to their base pairing properties, nucleicacid sequences can often form specific structures under certain solutionconditions. For example, in the presence of certain metal ions (e.g.,K⁺), short guanine (G)-rich sequences fold into a structure known as aG-quartet or quadruplex (see FIG. 3). Quadruplexes are very stable andbiophysical studies have shown that they possess intrinsic opticalproperties (e.g., absorb light at 300 nm) that distinguish them fromother secondary structures. Previously, quadruplex-formation assays havebeen developed, which exploit this unique quadruplex signature to studyenzymes that cleave DNA (Kankia, B. I. (2006) A real-time assay formonitoring nucleic acid cleavage by quadruplex formation, Nucleic acidsresearch, 34, e141) or facilitate strand-exchange reactions (Kankia, B.I. (2004) Optical absorption assay for strand-exchange reactions inunlabeled nucleic acids, Nucleic acids research, 32, e154). Briefly,when G-rich sequences with the potential to form a quadruplex areincorporated into DNA substrates they are initially in the quenchedstate. Upon enzymatic activity (i.e., strand cleavage orstrand-exchange) the released sequence folds into a quadruplex andbecomes visible when monitored by absorption or fluorescencespectroscopy.

One aspect of the present invention, then, uses the free energy of DNAquadruplexes (or other non-quadruplex conformations) to driveunfavorable (endergonic) reactions of nucleic acids (e.g., isothermalPCR). The key point of such reactions is that some sequences—e.g., someG-rich sequences—are capable of forming quadruplexes (or otherconformations) with significantly more favorable thermodynamics than thecorresponding DNA duplexes. The sequences are incorporated within DNAduplexes, which after interaction with an initiator (e.g., DNApolymerase) self-dissociate from the complementary strand and fold intoquadruplexes (or other conformations). The energy of formation of thenon B-DNA structure, or other DNA structure is used to drive PCR atsubstantially constant temperature.

Thus, in certain embodiments, the primer may include a sequence that isgenerally based on a sequence in the form of d(G₃₊N₁₋₇G₃₊N₁₋₇G₃₊N₁₋₇G₃)and include a label. In another embodiment, the primer may include asequence that is generally based on the (GGGT)₄ [SEQ. ID. NO. 1]sequence and includes a label such as 2Ap. And so, at least a portion ofthe primer sequence may have a sequence based on 2Ap-G3T(GGG2ApGGGTGGGTGGG) [SEQ. ID. NO. 10]. However, it will be recognized bythose of ordinary skill in the art that this sequence is not necessarilythe entire sequence of the primer, merely that the primer may includethe sequence based on 2Ap-G3T [SEQ. ID. NO. 10] as a portion of theoverall sequence of the primer, And so, this aspect of the presentinvention also provides a primer, wherein the portion of the primersequence based on 2AP-G3T [SEQ. ID. NO. 10] is attached to anothersequence specific for use as a primer to detect a desired target nucleicacid sequence.

And so, the problems with present nucleic acid amplification, detection,and quantification systems and methods are overcome by the presentlydescribed amplification process. Referring to FIG. 4, the generalprinciple of the amplification process is shown. The process usesspecific primers designed to fold into monomolecular quadruplexes uponelongation with significantly more favorable thermodynamics than thecorresponding DNA duplex. In particular, in the illustrated embodiment,a primer that is a truncated version of (GGGT)₄ [SEQ. ID. NO. 1] (a 13bprimer in the illustrated embodiment) and incorporates 2Ap is used. Inanother particular embodiment, this primer has the sequenceGGG(2Ap)GGGTGGGTGGG (2Ap-G3T) [SEQ. ID. NO. 10]. When this primer is notin the quadruplex conformation, fluorescence of 2Ap is quenched. Inalternate embodiments, the primer may include different, albeit similar,sequences. For example, in one alternate embodiment, the primer may havethe sequence GGG(2Ap)GGGTGGGTGG [SEQ. ID. NO. 11]. And in anotheralternate embodiment, the primer may have the sequence GGG(2Ap)GGGTGGGTG[SEQ. ID. NO. 12] (as in the illustrated embodiment). As can be seen inthe top panel of FIG. 4, before elongation, the primers (here shown as a13b primer e.g., GGG(2Ap)GGGTGGGTG [SEQ. ID. NO. 12]) form duplexes withthe target sequence since they are missing a few guanine residues thatwould result in quadruplex formation. Under PCR conditions, elongationthen begins, with the DNA polymerase adding dNTPs to the end of theprimers (as shown in the second panel of FIG. 4). This elongation theneventually adds the length and/or guanine residues necessary to allow aquadruplex structure to be formed. Once this occurs (see the third panelof FIG. 4), the 5′-end of each product DNA is trapped in a quadruplexand its complementary sequence (the target DNA) is fully accessible toanother incoming primer. And with the formation of the quadruplex, 2Apis no longer quenched. In still further embodiments, the primers mayhave the sequence GG(2Ap)TGGTGTGGTTGG [SEQ. ID. NO. 24] or may have thesequence GGTTGG(2Ap)GTGGTTGG [SEQ. ID. NO. 20]. Again, while thisprocess may be referred to herein as “QPA,” and while the schematic inFIG. 5 is described as showing “QPA,” those of ordinary skill in the artwill recognize that the aspects of the present invention are not limitedto primer sequences that form quadruplexes (as described previously).

Thus, the amplification process of the present invention has thepotential to expand the exponential growth phase of the reaction,because product self-annealing is eliminated due to the manner of thereaction (as shown in FIG. 4). As a result, the DNA yield can increasedramatically since each extra cycle will double the amount of productDNA. A longer exponential growth phase will also be very useful for DNAquantification by RT-PCR, as will be described in greater detail below.In addition, the amplification process using primers such as describedherein has the potential to eliminate non-specific products.Specifically, decreasing the annealing temperature can enhance productrefolding. This would reduce the availability of single-stranded regionsfor non-specific priming and further improve specific priming at targetsites constantly available for the primers.

Further, since the conformation taken on by the primer sequence (such asa quadruplex) is more stable than its corresponding duplex, unfolding ofthe duplex or release of target for the incoming primers can occurwithout the need of substantial temperature change or any temperaturechange. In other words, in standard PCR, following the extension step,the DNA is in a duplex form. The next cycle then begins by raising thetemperature to a point that the double-stranded DNA again denatures(i.e., separates into single strands). This is necessary in order toprovide the separated sense and anti-sense single-stranded DNA strandsfor primer binding (to each of the strands), followed by elongationduring the next extension step (once the temperature of the reaction isreduced). However, by using primers based on a sequence, such as the(GGGT)₄ [SEQ. ID. NO. 1] sequence, the primers and extending nucleotidesthat are added during the extension step naturally conform into astructure such as a quadruplex. As this occurs, the primer (e.g.,forming the quadruplex structure) naturally separates from the targetDNA sequence complementary to the primer, thereby leaving the targetregion complementary to the primer exposed in single-stranded form forbinding of the next primer. This occurs without requiring raising of thetemperature to denature the strands from one another. Thus,amplification can proceed under isothermal conditions. And so, theisothermal DNA amplification provided by the present invention does notrequire expensive instrumentation for thermocycling and may allow DNAamplification in the field and at point-of-care.

Thus, another aspect of the present invention provides an isothermicprocess for amplifying at least one target nucleic acid sequencecontained in a nucleic acid or a mixture of nucleic acids. The processincludes treating a nucleic acid or a mixture of nucleic acids with atleast one primer, such as an oligonucleotide primer, adapted to conforminto a structure that can dissociate from a DNA duplex structure in theabsence of heating [i.e., the primer includes a sequence that naturallyconforms into a structure, such as a quadruplex structure (or any othernon-B DNA configuration or other DNA structure) in which intramolecularbase pairing allows or causes the primer to dissociate from the doublestranded DNA—of which it is one strand—and form its particularstructure]. For example, the primer may be adapted to conform into aquadruplex structure during an extension step of a polymerase chainreaction, under isothermic conditions. During this isothermic process,for the at least one nucleic acid sequence being amplified, an extensionproduct of the at least one primer is synthesized which is complementaryto a strand from the nucleic acid or mixture of nucleic acids. In thisprocess, the at least one primer is selected so as to be sufficientlycomplementary to the strand from the nucleic acid or a mixture ofnucleic acids to hybridize therewith such that the extension productsynthesized from the at least one primer, when it is separated from itscomplement, can serve as a template for further synthesis of anextension product of another primer.

Thus, formation of the quadruplex, or other conformation, occursspontaneously in parallel to elongation and as a result, amplificationcan proceed under isothermal conditions. As such, isothermal DNAamplification does not require expensive instrumentation forthermocycling and allows DNA amplification in the field and atpoint-of-care. Several versions of isothermal DNA amplification havepreviously been developed [see Tomita, N. et al. (2008) Loop-mediatedisothermal amplification (LAMP) of gene sequences and simple visualdetection of products. Nature protocols, 3, 877-882; Vincent, M., et al.(2004) Helicase-dependent isothermal DNA amplification. EMBO reports, 5,795-800; Andras, S. C. et al. (2001) Strategies for signal amplificationin nucleic acid detection. Molecular biotechnology, 19, 29-44; Walker,G. T. et al. (1992) Strand displacement amplification—an isothermal, invitro DNA amplification technique. Nucleic acids research, 20,1691-1696; Walker, G. T. et al. (1992) Isothermal in vitro amplificationof DNA by a restriction enzyme/DNA polymerase system. Proceedings of theNational Academy of Sciences of the United States of America, 89,392-396; Fox, J. D. et al. (2009) In Logan, J. et al. (eds.), Real-timePCR. Caister Academic Press, Norfolk, UK, pp. 163-175]. However, allthese existing systems require extra reaction components or polymeraseswith specific activities, whereas QPA does not.

The process described above may further include separating the primerextension products from the templates on which they were synthesized toproduce single-stranded molecules. And, the process may further includetreating the single-stranded molecules with the at least one primerunder isothermic conditions such that a primer extension product issynthesized using each of the single strands as a template.

The primers used in this isothermic application may be the primersadapted to form quadruplexes, as described above. Thus, the primers maybe designed with a sequence having a G content of an amount (or toobtain a high enough amount) such that the primer conforms into aquadruplex structure during an extension step of PCR. In one embodiment,the G content of the sequence of a primer of this aspect of the presentinvention is equal to or greater than 70%. In another embodiment, the Gcontent may be equal to or greater than 75%. More specifically, in oneembodiment, the primer may be an oligonucleotide having a sequence basedon GGGTGGGTGGGTGGGT [SEQ. ID. NO. 1] [“(GGGT)₄” ]. However, it will berecognized by those of ordinary skill in the art that primers for use inthis aspect of the present invention do not have to include the exact(GGGT)₄ [SEQ. ID. NO. 1] sequence. As described above, other sequencesmay form quadruplexes provided they include a guanine amount that issufficient to form such quadruplexes. And, as described above, theprimers do not have to have sequences that form quadruplexes. Rather,any primer adapted to conform into a structure that can dissociate froma DNA duplex structure in the absence of heating will suffice for theprinciples of the present invention [i.e., the primer includes asequence that naturally conforms into a structure, such as a quadruplexstructure (or any other non-B DNA configuration or other DNA structure)in which intramolecular base pairing allows or causes the primer todissociate from the double stranded DNA—of which it is one strand—andform its particular structure].

Further, in order to be detectable, such as for use in RT-PCR, theprimer may have a label incorporated therein. Such a label may be chosenfrom labels that are known to those of ordinary skill in the art. Suchlabels include, but are not limited to, fluorescent labels. In oneparticular embodiment, the primer may have a fluorescent labelincorporated therein. And in a particular embodiment, such a label mayinclude 2Ap. However, other fluorescent nucleotides may includepteridine analogs: 3-methyl isoxanthopterin (3MI) (Ex348, Em431),6-methylisoxanthopterin (6MI) (Ex340, Em430) and(4-amino-6-methyl-8-(2¢-deoxy-â-D-ribofuranosyl)-7(8H)-pteridone (6AMP)(Ex330, Em435).

Thus, in certain embodiments, the primer may include a sequence that isgenerally based on the (GGGT)₄ [SEQ. ID. NO. 1] sequence and includes alabel such as 2Ap. And so, at least a portion of the primer sequence mayhave a sequence based on 2Ap-G3T [SEQ. ID. NO. 10]. For example, onesuch primer may have the sequence GGG2ApGGGTGGGTGGG (2Ap-G3T) [SEQ. ID.NO. 10]. However, it will be recognized by those of ordinary skill inthe art that this sequence is not necessarily the entire sequence of theprimer, merely that the primer may include the sequence based on 2Ap-G3T[SEQ. ID. NO. 10] as a portion of the overall sequence of the primer,And so, this aspect of the present invention also provides a primer,wherein the portion of the primer sequence based on 2Ap-G3T [SEQ. ID.NO. 10] is attached to another sequence specific for use as a primer todetect a desired target nucleic acid sequence.

Another unique property of the amplification process and primersdisclosed herein is that 2Ap incorporated in the primers results instrong fluorescence emission upon formation of a structure such as aquadruplex, as will be described in greater detail below, thus servingas a sensitive real-time detection probe.

Further, as described above, a drawback of current RT-PCR-specificquantification systems is that they use FRET-based applications, whichrequire costly synthesis and considerable effort to design a sensitiveprobe. A quantification mechanism that uses intrinsic fluorescence ofprimers will significantly simplify the detection process. And so,another aspect of the present invention provides a quadruplex formingsequence with incorporated 2Ap (2Ap-G3T) that emits strong fluorescenceupon formation of a structure such as a quadruplex. Thus, another aspectof the present invention provides a QPA-based real-time quantificationsystem (RT-QPA) using 2Ap as a sensitive probe for quantification.

More specifically, another aspect of the present invention provides areal-time quantification PCR method for detecting amplification of atarget nucleic acid. The method includes treating a nucleic acid or amixture of nucleic acids with at least one primer, such as anoligonucleotide primer, adapted to conform into a structure that candissociate from a DNA duplex structure in the absence of heating, suchas into a quadruplex structure during an extension step of anamplification reaction, such as RT-PCR polymerase chain reaction. In themethod, the at least one primer includes a label that is quenched whenthe at least one primer is in a non-dissociative conformation (like anon-quadruplex conformation) and that is detectable when the at leastone primer is in a dissociative conformation (like a quadruplexconformation).

Further, in order to be detectable, such as for use in RT-PCR, theprimer may have a label incorporated therein. Such a label may be chosenfrom labels that are known to those of ordinary skill in the art. Suchlabels include, but are not limited to, fluorescent labels. In oneparticular embodiment, the primer may have a fluorescent labelincorporated therein. And in a particular embodiment, such a label mayinclude 2Ap.

Thus, in certain embodiments, the primer may include a sequence that isgenerally based on the (GGGT)₄ [SEQ. ID. NO. 1] sequence and includes alabel such as 2Ap. And so, at least a portion of the primer sequence mayhave a sequence based on 2Ap-G3T [SEQ. ID. NO. 10]. For example, onesuch primer may have the sequence GGG2ApGGGTGGGTGGG (2Ap-G3T) [SEQ. ID.NO. 10]. However, it will be recognized by those of ordinary skill inthe art that this sequence is not necessarily the entire sequence of theprimer, merely that the primer may include the sequence based on 2Ap-G3T[SEQ. ID. NO. 10] as a portion of the overall sequence of the primer,And so, this aspect of the present invention also provides a primer,wherein the portion of the primer sequence based on 2Ap-G3T [SEQ. ID.NO. 10] is attached to another sequence specific for use as a primer todetect a desired target nucleic acid sequence. And, again, the G-richsequences described herein are merely exemplary.

Thus, after polymerase elongation, the specifically designedguanine-rich primers are capable of forming quadruplexes withsignificantly more favorable thermodynamics than the corresponding DNAduplexes. As a result, target sequences are always accessible for theprimers since their complementary strands are trapped in a dissociativeconformation (such as a quadruplex) and DNA amplification can proceedunder isothermal conditions. In addition, 2Ap nucleotides incorporatedand fully quenched within the primers regain maximum emission uponquadruplex formation allowing very simple and accurate detection ofproduct DNA. Thus, the amplification process described herein (i) lacksa product self-annealing; (ii) can proceed under isothermal conditions;and (iii) uses intrinsic fluorescence of primers for quantification ofDNA products.

Thus, to eliminate the drawbacks inherent in the presently usedmolecular beacons, TaqMan®, and Scorpion™ probes, the present inventionprovides, in one exemplary embodiment, QPA for use in RT-PCR (referredto herein as RT-QPA). Again, while “QPA” and “RT-QPA” is used herein,those of ordinary skill in the art will recognize that the amplificationprocess and primers are not limited to those that form quadruplexes—butinclude sequences that form any dissociative structures. RT-QPA is basedon fluorescence of 2Ap incorporated within the QPA primers. As describedabove, 2Ap is a fluorescent analog of adenine, which forms Watson-Crickbase-pairs with thymidine [Law, S. M. et al. (1996) Spectroscopic andcalorimetric characterizations of DNA duplexes containing 2-aminopurine.Biochemistry, 35, 12329-12337; McLaughlin, L. W. et al. (1988) A newapproach to the synthesis of a protected 2-aminopurine derivative andits incorporation into oligodeoxynucleotides containing the Eco RI andBam HI recognition sites. Nucleic Acids Res, 16, 5631-5644] and is welltolerated by DNA polymerases [Fidalgo da Silva, E. et al. (2002) Using2-aminopurine fluorescence to measure incorporation of incorrectnucleotides by wild type and mutant bacteriophage T4 DNA polymerases.The Journal of biological chemistry, 277, 40640-40649]. Free 2Ap has avery high quantum yield (0.68) which is ˜100-fold reduced uponincorporation into a DNA duplex [Ward, D. C. et al. (1969) Fluorescencestudies of nucleotides and polynucleotides. I. Formycin, 2-aminopurineriboside, 2,6-diaminopurine riboside, and their derivatives. J BiolChem, 244, 1228-1237]. Unfolding of the DNA duplex into complementarysingle-strands is typically accompanied by only a several-fold increasein fluorescence [Law, S. M. et al. (1996) Spectroscopic and calorimetriccharacterizations of DNA duplexes containing 2-aminopurine.Biochemistry, 35, 12329-12337; Menger, M. et al. (1996) Mg(2+)-dependentconformational changes in the hammerhead ribozyme. Biochemistry, 35,14710-14716; Rist, M. et al. (2001) Association of an RNA kissingcomplex analyzed using 2-aminopurine fluorescence. Nucleic Acids Res,29, 2401-2408]. Thus, the fluorescence of 2Ap present in single strandsis significantly quenched, which limits the sensitivity of the probe.Studies that developed the present invention show that the fluorescenceof 2Ap incorporated into the loop regions of a quadruplex displayfluorescence emission comparable to that of free 2Ap. Thus, RT-QPAprimers with intrinsic fluorescence represent simple and very sensitiveprobes for quantification of DNA amplicons, and have the potential toovercome the shortcomings of traditional detection methods discussedabove.

Thus, the advantages of RT-QPA are many. First, there is the reducedexpense of 2Ap containing primers relative to chemical synthesis ofseparate probes with dye-quencher pairs. Second, the primer-probe forany mRNA is universal. Third, since the detection probe is part of theprimer, QPA is free from complications introduced by separate probesequences. Fourth, the mechanism does not require special enzymaticactivity of a polymerase or heating steps. The detection can beperformed under isothermal conditions. Fifth, the primer is universalfor any PCR reaction, which completely eliminates the primer-probedesign step. And sixth, the mechanism is truly monomolecular whichguarantees immediate signal increase at the initial stage of theamplification process and efficient detection of early cycles invisiblein the case of bi-molecular detection mechanisms.

In addition, RT-QPA is extremely specific since specificity is notdetermined only by binding to a specific target, but also by properelongation of the next few bases to create a quadruplex. For instance,the reaction shown in FIG. 13 uses two cytidines (underlined) adjacentto the target sequence (bold type at 3′end). It is known thatquadruplexes can't tolerate nucleotide substitutions in G-tracts[Kankia, B. I. (2004) Optical absorption assay for strand-exchangereactions in unlabeled nucleic acids. Nucleic Acids Res, 32, p. 154].Thus, to produce a false signal, non-specific priming alone is notenough; the non-specifically bound primer would also have to bind atcytidine-tracts.

As will be appreciated by those of ordinary skill in the art,naturally-occurring target DNA will not necessarily include a sequencecomplementary to the primer sequence (e.g., will not necessarily includea sequence based on (GGGT)₄ [SEQ. ID. NO. 1] or other dissociativesequence). And so, the DNA templates need to have incorporated targetsequences, which can be accomplished by 2 cycles of traditional PCR. Theincorporation of the target sequence into a template is shownschematically in FIG. 5. The quadruplex folding sequence (dashed lines)will be attached at the 5′-end of both forward and reverse primers. Theproducts of the 2nd cycle (four duplexes at the end of PCR, FIG. 5)contain two single-stranded amplicons fully complementary to each otherwith incorporated target sites at the 3′-end (dotted lines). Thus, atthe end of the 2nd cycle, the number of amplicons with incorporatedtarget sites equals the initial amount of template, which is importantfor DNA quantification. At this point, the primer will be added to themix and amplification may be continued under isothermal cyclingconditions (although traditional thermal cycling conditions may also beused). Thereafter, the process will amplify only the desired shorttemplates, neglecting the other six unwanted templates which usually areamplified in traditional PCR.

Thus, to incorporate target sequences within templates, an extra step oftraditional PCR is required. However, difficulties at this step are notexpected, since only two cycles will be performed under favorableconditions for PCR exponential growth: high concentration ratio ofprimer over template. Moreover, as described above, one aspect of thepresent invention is that the entire process is isothermal. As usedherein, “isothermal” not only encompasses PCR that is truly isothermal(i.e., does not include any raising or lowering of temperatures, such asin a thermal cycle), but also encompasses PCR including an initial cycleor couple of cycles or few cycles that are used to incorporate thetarget sequences into the DNA template, as described above (and as shownschematically in FIG. 5, for example). In the case of RT-QPA, (i.e. geneexpression studies) no extra step is necessary since the target site canbe incorporated during cDNA synthesis.

The various aspects of the present invention will be described ingreater detail with respect to the following Examples.

Example 1

Example 1 describes preliminary studies that were designed and performedregarding the requirements of QPA and the design of the primers, todetermine the optical and thermodynamic properties of thequadruplex-forming sequence.

Requirements of QPA and Design of the Primers.

According to the QPA mechanism (FIG. 4), primers should dissociate froma target site and fold into a monomolecular quadruplex followingpolymerase elongation. To do so, the quadruplex should bethermodynamically more stable than the corresponding DNA duplex. At thesame time, the QPA mechanism also assumes that the primers (shorterversions of the quadruplex sequence) readily form duplexes with thetarget site (a prerequisite for DNA polymerase activity). It is alsodesirable that the sequences tolerate base substitutions of fluorescentanalogs and emit a fluorescence signal upon quadruplex formation forreal-time quantification of the product.

Thus, a successful QPA primer should comply with the followingrequirements: (i) bind to the target site, (ii) upon elongationdissociate from the target site and form a quadruplex, and (iii) emitlight during the structural rearrangement. And so, this Example focusedon the (GGGT)₄ [SEQ. ID. NO. 1] sequence which folds into a stablemonomolecular quadruplex in the presence of K⁺[as described in Jing, N.,Rando, R. F., Pommier, Y. and Hogan, M. E. (1997) Ion selective foldingof loop domains in a potent anti-HIV oligonucleotide, Biochemistry, 36,12498-12505, incorporated by reference herein in its entirety].

Optical and Thermodynamic Properties of Quadruplex Forming Sequence.

As described above, a unique property of QPA is that 2Ap incorporated inthe primers results in strong fluorescence emission upon quadruplexformation, thus serving as a sensitive real-time detection probe. UVmelting studies as a function of DNA strand concentration have shownthat (GGGT)₄ [SEQ. ID. NO. 1] folds into a monomolecular structure(T_(m) doesn't depend on strand concentration) that was unusuallystable. For instance, in the presence of 50 mM KCl and 2 mM MgCl₂, the(GGGT)₄ [SEQ. ID. NO. 1] quadruplex melts above 100° C. Removal of theterminal thymidine did not change the CD profile of the quadruplex (seeFIG. 6, -□-). Therefore, in further experiments, the truncated sequence(GGGT)₃GGG [SEQ. ID. NO. 2] was used. To establish appropriate positionsfor 2Ap, a (GGGT)₃GGG sequence with T→2Ap substitutions at the 4thposition [SEQ. ID. NO. 10] was also studied, which didn't reveal anychange in CD profile (see FIG. 6, -∘-), demonstrating that thequadruplex will tolerate 2Ap nucleotides at these positions [a(GGGT)₃GGG sequence with T→2Ap substitutions at the 4^(th) and 12^(th)positions was also studied, without any change in CD profile]. T→2Apsubstitution in the 4th position revealed a striking fluorescenceeffect: upon formation of GGG(2Ap)GGGTGGGTGGG (2Ap-G3T) [SEQ. ID. NO.10], the fluorescence emission of 2Ap reaches the level of its freestate (see FIG. 7).

Thus, in experiments, a construct with incorporated 2Ap as follows wasused: GGG(2Ap)GGGTGGGTGGG [“2Ap-G3T” ] [SEQ. ID. NO. 10],GGG(2Ap)GGGTGGGTGG [SEQ. ID. NO. 11] [“G3T-ss14” ], GGG(2Ap)GGGTGGGTG[SEQ. ID. NO. 12] [“G3T-ss13” ], GGG(2Ap)GGGTGGGTGGG [SEQ. ID. NO. 10]in duplex with its complementary strand CCCACCCACCCTCCC [SEQ. ID. NO. 3][“G3T-ds15” ], and GGG(2Ap)GGGTGGGTG [SEQ. ID. NO. 12] in duplex with acomplementary strand CCCACCCACCCTCCC [SEQ. ID. NO. 3].

Principle of QPA

Role of Cations and Terminal Guanines in Quadruplex Formation.

FIG. 8 demonstrates fluorescence unfolding experiments of G3T-ss15 [SEQ.ID. NO. 10], G3T-ss14 [SEQ. ID. NO. 11], and G35-ss13 [SEQ. ID. NO. 12].Unfolding of G3T-ss15 [SEQ. ID. NO. 10] was performed in the presence of50 mM monovalent cations, Na⁺ (-∘-), K⁺ (black line) and Cs⁺ (-•-). Inthe case of Na+ ions the melting curve reveals the sigmoidal behaviorcharacteristic of monophasic transition with T_(M)˜45° C. The transitioncorresponds to unfolding of the quadruplex, which is accompanied byquenching of 2Ap fluorescence by adjacent guanines in the unfoldedquadruplex. As expected [as shown by Jing, N., Rando, R. F., Pommier, Y.and Hogan, M. E. (1997) Ion selective folding of loop domains in apotent anti-HIV oligonucleotide, Biochemistry, 36, 12498-12505], thepotassium salt of G3T-ss15 is very stable with a T_(M) of ˜88° C. (blackline). Thus, both Na⁺ and K⁺ ions are able to fold quadruplexes, howeverthe latter is almost 45° C. more stable. In the presence of Cs⁺ ionsG3T-ss15 [SEQ. ID. NO. 10] does not reveal any measurable fluorescenceover the entire temperature range, which suggests that Cs⁺ does notsupport quadruplex formation [Kankia, B. I. and Marky, L. A. (2001)Folding of the thrombin aptamer into a G-quadruplex with Sr(2+):stability, heat, and hydration, Journal of the American ChemicalSociety, 123, 10799-10804]. The results are in agreement withobservations that K⁺ ions with ionic radii of 1.33 Å are the optimumsize for a cation to enter the inner core of G-quartets, while Cs⁺ ionswith ionic radii of 1.69 Å are too big [Jing, N., Rando, R. F., Pommier,Y. and Hogan, M. E. (1997) Ion selective folding of loop domains in apotent anti-HIV oligonucleotide, Biochemistry, 36, 12498-12505].

The role of terminal guanines in quadruplex formation in the presence ofK⁺ ions was studied similarly. Deletion of a single guanine at the3′-end, G3T-ss14 [SEQ. ID. NO. 11], significantly destabilized thequadruplex (FIG. 8, -Δ-). However, it is still able to create somestructure at lower temperatures. Deletion of another guanine, G3T-ss13[SEQ. ID. NO. 12], almost completely inhibits quadruplex formation(-□-). Thus, the experiments shown in FIG. 8 suggest that (i) in thepresence of Cs⁺ ions mixing of full-length G3T-ss15 [SEQ. ID. NO. 10] toits complementary sequence should result in a DNA duplex; and (ii) inthe presence of K⁺ ions the truncated variant, G3T-ss13 [SEQ. ID. NO.12], should also be able to form a duplex.

Role of Cations and Terminal Guanines in Duplex Formation.

To mimic DNA conformational changes that take place upon the QPAreaction, the fluorescence melting of the G3T-ds15 [SEQ. ID. NO. 10]duplex was studied in amplification buffer (15 mM KCl, 35 mM CsCl, 2 mMMgCl₂, 10 mM Tris-HCl, pH 8.7) (FIG. 9). To ensure that G3T-ds15 [SEQ.ID. NO. 10] initially anneals to its complementary strand (asdouble-helix), the sequences were annealed in the presence of CsClfollowed by later KCl addition. (K⁺ is a quadruplex forming cation,while Cs⁺ does not support quadruplexes [as described in Kankia, B. I.et al. (2001) Folding of the thrombin aptamer into a G-quadruplex withSr(2+): stability, heat, and hydration, J Am Chem Soc, 123, 10799-10804,incorporated by reference herein in its entirety.]). In QPA, the duplexis formed by annealing a shorter version of 2Ap-G3T [SEQ. ID. NO. 10](unable to form a quadruplex) to the target sequence with subsequentaddition of the missing bases by Taq polymerization. The heating curve(black curve, FIGS. 9 and 11) reveals two separate transitions withmidpoints at 60° C. and ˜95° C. The transition at 60° C. corresponds toduplex unfolding, which is accompanied by an increase in fluorescencedue to quadruplex formation of released G3T-ss15 [SEQ. ID. NO. 10]. Thesecond transition at 95° C. corresponds to the melting of the quadruplexaccompanied by fluorescence quenching of 2Ap due to stackinginteractions of adjacent guanines in unstructured 2Ap-G3T [SEQ. ID. NO.10]. The second transition is completely reversible during the coolingprocess (-□- in FIGS. 9 and 11). However, no duplex refolding wasobserved, which clearly indicates that the quadruplex stays folded atlower temperatures in the presence of the complementary strand. Inseparate isothermal experiments at 40° C., the complementary strand wasadded to a preformed G3T-ss15 quadruplex [SEQ. ID. NO. 10], which didn'taffect the fluorescence spectrum of the quadruplex (data not shown).Thus, both melting and isothermal mixing experiments show that thequadruplex is very stable and the complementary strand is unable toinvade the structure. Taken together, these data indicate that G3Tprimers can potentially inhibit product DNA re-annealing at targetsites.

It is noted that the duplex melting temperature (˜60° C.) measured inthe presence of the quadruplex forming cation KCl (FIGS. 9 and 11) issignificantly lower than the T_(M)=70° C. of the same duplex measuredunder experimental conditions unfavorable for quadruplex formation (50mM CsCl and 2 mM MgCl₂), or predicted from nearest-neighbor analysis ofequilibrium unfolding [Zuker, M. (2003) Mfold web server for nucleicacid folding and hybridization prediction, Nucleic acids research, 31,3406-3415]. To compare thermal stabilities of the G3T-ds15 [SEQ. ID. NO.10] duplex in the presence and absence of K⁺, UV absorption was employed(FIG. 10). In the presence of K⁺ ions G3T-ds15 [SEQ. ID. NO. 10] unfoldsat 60° C. (-∘-), which is in excellent agreement with results of thefluorescence measurements shown in FIG. 9. In the absence of K⁺ ions theduplex is significantly more stable and unfolds at 70° C. (-□-) aspredicted from nearest-neighbor analysis of equilibrium unfolding[Zuker, M. (2003) Mfold web server for nucleic acid folding andhybridization prediction, Nucleic acids research, 31, 3406-3415]. Notean additional small peak at 93° C. in the presence of K⁺, whichcorresponds to quadruplex unfolding and again agrees with fluorescencemeasurements shown in FIG. 9. Additional melting experiments of theG3T-ds15 [SEQ. ID. NO. 10] duplex in the presence of K⁺ performed atslower heating rates (0.5° C./min and 0.1° C./min) further shifted thetransition to lower temperatures (data not shown). Thus, in the presenceof K⁺, unfolding of the duplex is a non-equilibrium process due toquadruplex formation of the released strands, which significantlydestabilizes the duplex. FIG. 10 also demonstrates unfolding of G3T-ds13[SEQ. ID. NO. 12] in the presence and absence of K⁺ ions. Since G3T-ss13[SEQ. ID. NO. 12] is not able to form a quadruplex (see FIG. 8),G3T-ds13 [SEQ. ID. NO. 12] duplex melting profiles are identical in thepresence and absence of K⁺ ions with T_(M)=65° C. As expected, in thepresence of Cs⁺ ions the longer duplex, G3T-ds15 [SEQ. ID. NO. 10], ismore stable than the shorter duplex, G3T-ds13 [SEQ. ID. NO. 12].However, in the presence of K⁺ the opposite is true: the shorter duplexis ˜5° C. more stable than the longer one. This result illustrates thepotential for isothermal QPA; at appropriate temperatures, the primer ismore stable before elongation, which facilitates primer dissociation andthe next priming round without the need for thermal denaturation.

Optical and Thermodynamic Properties of the Primers.

As mentioned earlier, QPA primers, which in this Example 1 are truncatedversions of G3T, should bind target sequences and form duplexes.Referring to FIG. 12, deletion of a single guanine at the 3′-end of G3Tsignificantly destabilized the quadruplex as expected (-∘-), however, itis still able to create some quadruplex structure at lower temperatures.Deletion of another guanine completely inhibits quadruplex formation(-Δ-). Thus, the 13-base long GGG(2Ap)GGGTGGGTG [SEQ. ID. NO. 12]sequence fully meets primer requirements as: (i) it binds to the targetsequence; (ii) upon addition of two bases it can fold into a quadruplex,and (iii) quadruplex formation is accompanied by a strong increase in2Ap fluorescence.

QPA Under Isothermal Conditions.

As described above, since the quadruplex is more stable than itscorresponding duplex, unfolding of the duplex can proceed without theneed for temperature change. To demonstrate this potential of isothermalQPA, and referring now to FIG. 13, a chemically synthesized 51-base longtemplate with an incorporated target site at the 3′-end (bold type) anda quadruplex forming sequence at the 5′-end (bold type) was used. Thus,the template mimics one strand of a DNA amplicon obtained afterincorporation of a target site in a DNA template (described in FIG. 5).QPA reactions were performed under experimental conditions (shown inFIG. 11) at 55° C. The experimental temperature was predetermined by theT_(m) of the primer-target duplex, which is 62° C. The level offluorescence signal achieved at the plateau region corresponds to 0.8-1μM quadruplex, which indicates that most of the primers were used in theelongation reactions.

Summary of Data Obtained from Example 1.

Example 1 demonstrates that 2Ap incorporated and quenched within the G3Tsequence regains its maximum emission upon quadruplex formation. Thefluorescence is comparable to the level of free 2Ap base and sensitiveenough to monitor DNA amplification in real-time. Since 2Ap is anintrinsic part of the primers, QPA offers a very simple, inexpensive andtruly single-molecular primer/probe system with high sensitivity. Thus,QPA has the potential to overcome most of the shortcomings of currentquantitative PCR-based detection mechanisms. The G3T-quadruplex staysfolded in the presence of the complementary strand. As a result, the5′-end of each product DNA is trapped in a quadruplex and itscomplementary sequence (target) is fully accessible to the next round ofprimers. Thus, QPA has the potential to liberate traditional PCR fromproblems associated with product self-annealing, and improve itsefficiency. Example 1 also demonstrates that QPA can proceedspontaneously under substantially isothermal conditions.

Example 2

To further demonstrate the potential of isothermal QPA, two similartemplates with incorporated primer binding sites (PBS) (bold typesegments at 3′-end in FIG. 14, panel A) were used. The “exponential”template contains a quadruplex forming sequence at the 5′-end (bold typesegment at 5′end in FIG. 14, panel A), which is fully complementary to19-base PBS and allows incorporation of the PBS to newly generatedamplicons (FIG. 14, panel B). The amplicons are used as templates in thefollowing rounds of replication, setting up an exponential amplificationpattern. In the “linear” template, the quadruplex forming sequence isscrambled. As a result, Taq is able to replicate only the initialtemplates, setting up a linear amplification pattern (FIG. 14, panel C).

Further, the primer having a GCGC sequence at the 5′-end, whichincreases the thermal stability of primer-target complex to 76° C. andallows isothermal QPA to be performed at optimal temperatures forenzymatic activity of Taq (70-75° C.). To ensure that the attachment ofGCGC does not affect the quadruplex forming ability of G3T-ss15 [SEQ.ID. NO. 10], additional studies were performed (data not shown), and nosignificant effects of GCGC-attachment was observed. Additionally, inthe exponential template, PBS and quadruplex-forming segments are fullycomplementary to each other with a possibility of forming a stem, thusinhibiting primer binding. However, in the presence of K⁺ ions, thequadruplex sequence folds into a quadruplex and the PBS is accessiblefor primers, as shown schematically in FIG. 14B.

Experiments at 100 nM Template.

Experiments have been designed to demonstrate the isothermal nature ofQPA at rather high concentrations of template (100 nM). The addition ofTaq polymerase into reaction mixtures initiated rapid fluorescenceemission (FIG. 15), which is attributed to quadruplex formation uponpolymerase elongation. Results of these experiments reveal three majorfeatures of QPA: (i) amplification is isothermal without any input ofadditional factors or enzymes; (ii) DNA yield appears to be unusuallyhigh and reaches the initial concentration of primers, 1 μM (traditionalPCR usually plateaus at low nanomolar concentrations); and (iii)amplification can be monitored by intrinsic fluorescence of primerscontaining 2Ap. To confirm that the fluorescence increase in FIG. 15Acorresponds only to quadruplex formation, negative controls in theabsence of (i) quadruplex forming cations, (ii) Taq, and (iii) templateswere performed, which did not reveal any fluorescence increase (data notshown).

As shown in FIG. 15A, increasing reaction temperature results in QPArate enhancement, however, at higher temperatures product yielddeclines. For instance, at 70° C. QPA reaches 100% yield at ˜50 min,while at 72° C. the reaction levels off at ˜30 min with 80% yield. Therole of temperature is not clear from the preliminary experiments, sinceQPA includes several steps (priming, elongation and dissociation ofelongated primer from PBS). Each of these processes strongly depends onthe temperature as well as the ionic strength. Thus, the role oftemperature and ionic strength to find out the most favorable conditionsfor QPA will be studied.

Comparison of the fluorescence results in FIG. 15A with gelelectrophoresis of QPA products in FIG. 15B also reveals that QPAdetection is superior at monitoring amplification of specific productsonly. Lanes 5 and 6 correspond to products formed at 70 min and afterovernight reaction (10 h), respectively. Note that the overnightincubation produced higher molecular weight DNA, which is likely theresult of Taq enzymatic activity after consuming the primers. Howeverthe non-specific DNA, which is detected by electrophoresis isundetectable by fluorescence measurements (plateau level stays the sameduring the overnight reaction, data not shown). The high accuracy ofQPA, or sensitivity to only specific products, can be explained by thefact that its specificity is not only determined by target binding, butalso by proper elongation of the next few bases to create a quadruplex.For instance, the QPA described in FIG. 15A requires two cytidines(underlined in FIG. 14A) adjacent to the PBS. It is known thatquadruplexes do not tolerate nucleotide substitutions in their G-tracts[Kankia, B. I. (2004) Optical absorption assay for strand-exchangereactions in unlabeled nucleic acids, Nucleic acids research, 32, p.154; Smirnov, I. and Shafer, R. H. (2000) Effect of loop sequence andsize on DNA aptamer stability, Biochemistry, 39, 1462-1468]. Thus,nonspecific priming alone is not sufficient to form a quadruplex andproduce a false signal. In addition, the non-specifically bound primerwould have to bind at cytidine tracts, which further decreases theprobability of a false signal.

Experiments at Low Concentrations of Templates.

The experiments shown in FIG. 15, were performed under experimentalconditions where Taq is the limiting reagent. Thus, they do not revealthe amplification potential (exponential or linear) of QPA. In order toperform exponential amplification, Taq polymerase must replicate theentire template including the quadruplex (FIG. 14). To do so, Taq mustunfold the G3T-ss15 [SEQ. ID. NO. 10] quadruplex upon polymerization,which may be impeded by the high stability of the quadruplex. Thus, highconcentrations of K⁺ can have both positive (accelerate QPA throughfaster primer-PBS dissociation) and negative effects (limit QPA to onlylinear amplification). Thus, optimizing K⁺ concentration will be acritical first step to find optimal conditions for exponentialamplification by QPA. In a preliminary study, both processes werefavored (primer-PBS dissociation and quadruplex unfolding/replication byTaq) by performing thermo-cycling QPA at low concentrations of K⁺ ions(5 mM). Typical thermo-cycling QPA reactions conducted at 10 fM templateare shown in FIG. 16A. In the presence of the exponential template (seeFIG. 14A), clear exponential increase of the fluorescence signal afterthe 17th cycle was observed. QPA points from 17 to 26 in FIG. 16A werefit to the exponential expression to obtain the slope, and to calculatethe efficiency (E) of QPA, E=e^(slope)=e^(0.62525)=1.87 (E=2 correspondsto 100% efficiency). As expected, QPA using a linear template does notshow any significant fluorescence increase (squares, FIG. 16A). Todetect measurable effects for both exponential and linear amplificationunder the same conditions, similar experiments were performed in thepresence of 1 nM templates (FIG. 16B). In this reaction, Taqconcentration becomes limiting after a few cycles, however, thedifference between linear and exponential amplification is stillobvious.

To reveal the exponential nature of QPA under isothermal conditions,different experimental conditions were tested, and good activity wasobserved using 10 pM template in the presence of 25 mM KCl at 72° C.(FIG. 17A). The fluorescence signal increases after 40 min and at ˜100min reaches a plateau corresponding to ˜0.8 μM single-stranded product.The reaction was allowed to proceed for another 10 hours, and duringthis time the fluorescence signal remained at the plateau level. Productformation was also followed by agarose gel electrophoresis (FIG. 17B).These results demonstrate that non-specific DNA starts to accumulateafter the plateau is reached. This product does not have any significanteffect on the fluorescence signal (FIG. 17A), most likely because itdoes not involve the quadruplex forming sequence used to detect thedesired product. Thus, this experiment confirms that QPA detection issensitive to only the specific product even in the presence ofsignificant amounts of non-specific DNA.

Incorporation of QPA Primer Binding Sites into DNA Templates.

Since QPA uses a universal primer, the PBS must be incorporated into atarget template. This can readily be accomplished by only two cycles oftraditional PCR in which the 5′-end of both forward and reverse primershave quadruplex attachments (FIG. 5). Note that here the quadruplexattachments are used only for PBS incorporation, and not for 2Ap-baseddetection. The product of the 2nd PCR cycle contains two amplicons withincorporated target sites at the 3′-end (FIG. 5, dashed lines). Thus, atthe end of the 2nd cycle, the number of amplicons with incorporated QPAtarget sites equals the initial amount of template, which is importantfor accurate DNA quantification. QPA will amplify only these two desiredtemplates, and not the other six unwanted templates, which are usuallyamplified in traditional PCR.

FIG. 18 demonstrates successful QPA target site incorporation into apUC18 cloning region using the sequencing primers (forward primer,GTAAAACGACGGCCAGT [SEQ. ID. NO. 4], T_(M) of 64° C.; reverse primer,GGAAACAGCTATGACCA [SEQ. ID. NO. 5], T_(M) of 59° C.) with an attachedquadruplex-forming sequence (GCGC-G3T-ss15) [SEQ. ID. NO. 22]. Initialtwo cycles of traditional PCR were conducted at three differenttemperatures (53° C., 72° C. and 94° C. for 30 s each). Then, theQPA-primer was added and RT-QPA was performed by cycling between twotemperatures as follows: 72° C. for 60 s and 94° C. for 12 s, whichdemonstrates exponential amplification (FIG. 18). Note that 72° C. istoo high for the sequencing primers and their priming is negligibleduring QPA. As expected, the negative control (no initial sequencingprimers) shows no amplification (squares, FIG. 18). While a fewtemperature changes can be easily performed in a laboratory without athermocycler, truly isothermal systems are desirable for Global Healthdiagnostics. However, even semi-isothermal QPA described in FIGS. 5 and18 will be a very useful addition to any PCR-based detection platforms,since its high yield will allow thorough multi-well diagnostics.

Example 3

To further demonstrate the potential of QPA, a chemically synthesized 59base long template with an incorporated target site at the 3′ end (FIG.19, -∘-) and a quadruplex-forming sequence at the 5′ end (FIG. 19, -Δ-)was used. This template mimics one strand of a DNA amplicon obtainedafter incorporation of a target site in a DNA template (as describedpreviously, and as shown schematically in FIG. 5). The primer has theGCGC sequence attachment at its 5′ end, which increases its T_(m) to 76°C. [as described in Zuker, M. Mfold web server for nucleic acid foldingand hybridization prediction, Nucleic Acids, 31, pp. 3406-3415 (2003)],and allows isothermal QPA to be performed at optimal temperatures forTaq activity (70-75° C.). QPA was successfully performed underisothermal conditions.

Example 4

Example 4 is a prophetic example for the optimization of QPA. Inparticular, Example 4 will investigate efficiency of QPA at varyingconcentrations of KCl, primers, dNTPs and Taq polymerase. These studieswill define optimal conditions for QPA and for incorporation of QPAtarget sites into templates. It is expected that QPA will increase theexponential growth phase and increase DNA yield, thereby improving theefficiency of PCR.

Optimizing Experimental Conditions for QPA.

K⁺ is a quadruplex folding cation [Kankia, B. I. et al. (2001) Foldingof the thrombin aptamer into a G-quadruplex with Sr(2+): stability,heat, and hydration. J Am Chem Soc, 123, 10799-10804] and therefore itis an essential component of QPA (see FIG. 2). Increasing K⁺concentration will facilitate quadruplex formation and may accelerateQPA. This is due to the fact that the stability of the G3T quadruplex isproportional to the K⁺ concentration. However, high concentrations of K⁺can have negative effects on QPA efficiency. Specifically, in order toperform exponential amplification, Taq polymerase should replicate theentire template including the G3T quadruplex (see FIG. 5). To do so, Taqmust unfold the G3T quadruplex upon polymerization, which may be impededby the high stability of the quadruplex. Thus, high concentrations of K⁺can have both positive (accelerate QPA) and negative effects (limit QPAto only linear amplification). Therefore, it will be important tooptimize the concentration of K⁺ to achieve maximal QPA efficiency andthe longest exponential growth step.

Thus, RT-QPA will be performed at different concentrations of K⁺, whilemaintaining an ionic strength of 50 mM by adding appropriate amounts ofCsCl. Cs⁺ does not support quadruplex formation and acts as anon-specific cation to stabilize primer-target duplexes. Chemicallysynthesized templates (similar to those shown in FIG. 13), will be used,with the target sites corresponding to the most efficient primers, aswill be discussed below in Example 5.

In these reactions, QPA will be performed at femtomolar or picomolarconcentrations of the template. It is expected that exponentialamplification will result in an exponential increase in the fluorescencesignal. (At high concentrations of template a potential limiting factorcould be the amount of Taq rather than template concentration). As anegative control, a similar template without a quadruplex formingsequence at the 5′-end will be used.

In these reactions, newly produced DNA will be missing target sites andTaq can use only initial templates for amplification and one shouldobserve a linear increase in fluorescence measurements. Standardexperimental conditions for successful PCR typically include 0.1-1 μMprimers, 200 μM dNTPs and 1-5 Units of Taq. Since in QPA primerdimerization is excluded (QPA uses a single primer), complications athigher primer concentrations (i.e. 2-5 μM) are not expected. Inaddition, QPA has the potential to elongate the exponential part oftraditional PCR, and therefore increase the number of cyclescorresponding to the plateau region. Thus, testing QPA at varyingconcentrations of primers, dNTPs and Taq polymerase will be performed tofind the most efficient conditions.

If exponential growth at any concentration of K⁺, which means that Taqwas unable to invade stable K-quadruplexes, fails to occur, othercations known to form less stable quadruplexes (i.e. Rb+, NH4+, Na+ orBa2+) will be tested [Kankia, B. I. et al. (2001) Folding of thethrombin aptamer into a G-quadruplex with Sr(2+): stability, heat, andhydration. J Am Chem Soc, 123, 10799-10804].

Validating QPA as a More Efficient Amplification Method.

Incorporation of target sites into a DNA template will initially bedemonstrated on the commonly used pUC18 plasmid vector. To amplify the103-bp long multiple cloning site, the following standard 17-mersequencing primers will be used: forward primer, GTAAAACGACGGCCAGT [SEQ.ID. NO. 4] with a T_(m) of 64° C. and reverse primer, CAGGAAACAGCTATGAC[SEQ. ID. NO. 6], with a T_(m) of 58° C. in 50 mM monovalent cation and2 mM MgCl₂. The quadruplex forming sequence (i.e.,5′-CGGCGGGAGGGTGGGTGGG-3′) [SEQ. ID. NO. 7] will be attached at the5′-end of each primer.

Since only two cycles of traditional PCR are needed, combined 34-merprimers will be used at relatively low concentrations (10-50 nM). ThePCR will be performed at three different temperatures: 53° C. (priming),72° C. (elongation) and 94° C. (unfolding). After two cycles, the amountof DNA amplicons with incorporated target sites(5′-CCCACCCACCCTCCCGCCG-3′) [SEQ. ID. NO. 8] will be equal to theinitial number of DNA templates (see FIG. 5) and amplification will becontinued by QPA at 72° C. after adding 1 μM QPA primer(5′-CGGCGGG(2Ap)GGGTGGGTG-3′ [SEQ. ID. NO. 23] with T_(m) of ˜76° C.).Further, this temperature is too high for the initial 34-mer primers andtherefore their priming will be completely excluded. As a negativecontrol, the same reaction without initial primers will be performed.

Following the initial 2 cycles of PCR to incorporate the target sites,QPA vs. traditional PCR amplification strategies will be compared.Reactions will be performed using the same sequencing primers with andwithout quadruplex attachments. The amount of DNA in both systems (QPAwith 34-mer primers and PCR with 17-mer primers) following 30-40 roundsof amplification will be quantified using a molecular beacon with a 6-bplong stem and 20-nt long loop complementary to the product DNA. Thefluorescence will be monitored using an Eclipse spectrophotometer(Varian) with temperature controlled cell holders. Experiments will beperformed directly in fluorescence cells, and temperature will becontrolled automatically by the Eclipse software, which allows anunlimited number of temperature steps during a single experiment. It isexpected that the exponential growth phase for QPA will be longer thanfor traditional PCR.

Alternative Strategies.

All of these experiments are planned for using standard (multi-use)fluorescence cells. This will increase chances of contamination.Therefore, special care will be taken for cleaning the cells.Specifically, negative controls (no templates) will be routinelyperformed and appropriate washing procedure will be designed forcomplete eliminating of DNA products (i.e. using strong detergents, basesolutions or other DNA degrading agents).

Example 5

Example 5 is a prophetic example for establishing optimal experimentalconditions for isothermal QPA. Isothermal QPA will significantlysimplify current amplification systems since it does not requireexpensive instrumentation for thermocycling, or additional enzymes andallows DNA amplification in the field and at point-of-care.

Optimizing Primer Design for Isothermal QPA.

In our preliminary studies, a G3T sequence with a T_(m) of 62° C. in 50mM monovalent salt and 2 mM MgCl₂ was used, which predetermined therather low elongation step temperature of 55° C. (FIG. 11). As mentionedearlier, the most suitable temperature for isothermal QPA is 75° C.,which corresponds to Taq's optimal activity. In addition, at highertemperatures product duplexes will be more destabilized, which willfacilitate the strand-displacement process upon DNA elongation.Therefore, QPA experiments between 70° C. and 85° C. will be performed,and for each temperature new primers with corresponding T_(m)s will bedesigned. To increase the T_(m) of the primers, extra nucleotides willbe added at the 5′-end of G3T sequence. For instance, theCGGC-GGG(2Ap)GGGTGGGTG (CGGC-G3T) [SEQ. ID. NO. 23] primer demonstratesa T_(m) of 76° C. [Zuker, M. (2003) Mfold web server for nucleic acidfolding and hybridization prediction. Nucleic Acids Res, 31, 3406-3415],which will allow isothermal QPA to be performed at 70-72° C.

Design of Isothermal QPA.

As mentioned above, the tested version of QPA requires temperaturechanges during the initial two cycles (see FIG. 5), which allowsincorporation of QPA-target sites into the templates. While a fewtemperature changes can be easily performed in a laboratory without athermocycler, truly isothermal QPA is desirable during detection ofpathogenic microorganisms in the field, at check-points orpoint-of-care. Truly isothermal QPA will be designed, which is based onthe fact that QPA-target site incorporation occurs at very lowconcentration of the DNA templates (femtomolar or low picomolar). Atthese concentrations the stability of DNA polymers is greatly decreasedand they unfold at ˜85° C. in the presence of the 50 mM monovalentcations and 2 mM MgCl₂ [Zuker, M. (2003) Mfold web server for nucleicacid folding and hybridization prediction. Nucleic Acids Res, 31,3406-3415]. The thermal stability is further decreased to 82° C. in thepresence of 10 mM monovalent cations and 1 mM MgCl₂. It is hypothesizedthat by designing initial and universal primers with T_(m)s˜82-85° C.,one should be able to perform entire QPA under isothermal conditions.

To perform isothermal QPA at 80° C., a primer with a T_(m) of ˜84° C.will be designed, which would require a 7-9 nucleotide attachment to theG3T primer. Thus, the longer primers can potentially fold into dimers.Therefore, particular care will be taken during the primer design, andadditional experiments (CD spectroscopy and UV thermal unfolding) willbe performed to avoid primer dimerization.

Example 6

Example 6 is a prophetic example whereby an ultra-sensitive primer-probewill be designed by incorporation several 2Aps in QPA primers. DNA willbe quantified as a function of number of cycles (thermocycling QPA) andtime (isothermal QPA). These studies will develop a sensitive, robust,simple and universal real-time detection and quantification method whichwill significantly simplify current techniques.

Primers with Higher Fluorescence Yield.

As shown in the preliminary studies, 2Ap incorporated in the 4thposition of G3T gives ˜100-fold increase in fluorescence signal uponquadruplex formation (FIG. 7). Specifically, emission of 2Apincorporated within the folded quadruplex corresponds to emission of the2Ap free base with a quantum yield of 0.68 [Ward, D. C. et al. (1969)Fluorescence studies of nucleotides and polynucleotides. I. Formycin,2-aminopurine riboside, 2,6-diaminopurine riboside, and theirderivatives. J Biol Chem, 244, 1228-1237]. Thus, the fluorescence signalemitted upon QPA is sensitive enough to be used for product detection inRT-PCR. However, even more sensitive probes are proposed byincorporating several 2Aps at loop regions of G3T. The loop region isthe most convenient location to avoid quenching from neighboring basesand reach the maximum emission of 2Aps.

The reported NMR structure of (GGGT)₄ [SEQ. ID. NO. 1] [Jing, N. et al.(1998) Structure-activity of tetrad-forming oligonucleotides as a potentanti-HIV therapeutic drug. J Biol Chem, 273, 34992-34999] suggests thatthe quadruplex structure is monomolecular with opposite directions ofG-tracts (anti-parallel conformation) (FIG. 9A). However, CD spectracollected by us and published [in Jing, N. et al. (2001)Structure-activity of inhibition of HIV-1 integrase and virusreplication by G-quartet oligonucleotides. DNA Cell Biol, 20, 499-508incorporated by reference herein in its entirety] suggest that allstrands in the structure must be parallel [Lu, M. et al. (1993)Thermodynamics of G-tetraplex formation by telomeric DNAs. Biochemistry,32, 598-601]. Previously performed UV melting experiments also agreethat the structure is monomolecular and unusually stable [see Jing, N.,et al. (1997) Ion selective folding of loop domains in a potent anti-HIVoligonucleotide. Biochemistry, 36, 12498-12505 incorporated by referencein its entirety]. Based on the thermal stability of (GGGT)₄, CDcharacteristics, and the monomolecular nature of the complex, it ishypothesized that three G-quartets are formed by diagonal single T-loops(FIG. 20, panel B). Our model is supported by the fact that deletion ofthe terminal T16, which is not involved in the parallel structure, didnot reveal any destabilization (FIG. 6). In contrast, according to theanti-parallel conformation (FIG. 20, panel A), T16 is involved inquadruplex formation, and therefore its deletion should have asignificant destabilization effect.

Incorporation of 2Ap at the 4th position fully restored its emissionupon quadruplex formation (FIG. 7). The magnitude of the fluorescenceeffect can be explained by the fact that both adjacent guanines areimmobilized in stable G-quartets without any possibility of quenchingthe fluorophore (FIG. 20, panel B). Since T8 and T12 form similarsingle-nucleotide loops, testing 2Ap in these positions is proposed todesign an ultra-sensitive primer-probe of RT-QPA.

Alternative Strategies.

The hypothetical model of the parallel structure shown in FIG. 20, panelB is based on thermodynamic and spectroscopic studies. Three G-quartetswere assumed because of higher thermal stability of the G3T quadruplexwhen compared with the quadruplexes with two G-quartets [Kankia, B. I.et al. (2001) Folding of the thrombin aptamer into a G-quadruplex withSr(2+): stability, heat, and hydration. J Am Chem Soc, 123, 10799-10804;Hardin, C. C. et al. (2000) Thermodynamic and kinetic characterizationof the dissociation and assembly of quadruplex nucleic acids.Biopolymers, 56, 147-194 incorporated by reference herein in theirentireties]. However, one can't exclude two G-quartets in the G3Tsequence with three diagonal GT loops. To test this possibility,substitution at positions 3, 7 and 11 will be made and studied for theireffect on quadruplex formation. Depending on the outcome, incorporationof 2Ap in positions 3, 7 and 11 will also be tested.

Thus, the sensitivity of QPA probes may be further increased byincorporating more than one 2Ap within the loop regions of G3T-ss13[SEQ. ID. NO. 12]. It is believed that the loop region is the mostconvenient location to avoid quenching from neighboring bases and reachthe maximum emission of 2Ap. The reported NMR structure of (GGGT)₄ [SEQ.ID. NO. 1] [Jing, N. and Hogan, M. E. (1998) Structure-activity oftetrad-forming oligonucleotides as a potent anti-HIV therapeutic drug.The Journal of biological chemistry, 273, 34992-34999] suggests that thequadruplex structure is monomolecular with opposite directions ofG-tracts (anti-parallel conformation) (FIG. 20, panel A). However, CDspectra collected by us (data not shown) and published earlier [Jing,N., Marchand, C., Guan, Y., Liu, J., Pallansch, L., Lackman-Smith, C.,De Clercq, E. and Pommier, Y. (2001) Structure-activity of inhibition ofHIV-1 integrase and virus replication by G-quartet oligonucleotides. DNACell Biol, 20, 499-508] suggest that all strands in the structure mustbe parallel [Lu, M., Guo, Q. and Kallenbach, N. R. (1993) Thermodynamicsof G-tetraplex formation by telomeric DNAs. Biochemistry, 32, 598-601].UV melting experiments (not shown) also suggest that the structure ismonomolecular and unusually stable [Jing, N., Rando, R. F., Pommier, Y.and Hogan, M. E. (1997) Ion selective folding of loop domains in apotent anti-HIV oligonucleotide. Biochemistry, 36, 12498-12505]. Basedon the thermal stability of (GGGT)₄ [SEQ. ID. NO. 1], CDcharacteristics, and the monomolecular nature of the complex, it isbelieved that three G-quartets are formed by diagonal single T-loops(FIG. 20, panel B). This model is supported by the fact that deletion ofthe terminal T16, which is not involved in the parallel structure (FIG.20, panel B), did not reveal any destabilization (data not shown). Incontrast, according to the anti-parallel conformation (FIG. 20, panelA), T16 is involved in quadruplex formation, and therefore its deletionshould have a significant destabilization effect. As shown in FIG. 7,incorporation of 2Ap at the 4th position fully restored its emissionupon quadruplex formation. The magnitude of the fluorescence effect canbe explained by the fact that both adjacent guanines are immobilized instable G-quartets without any possibility of quenching the fluorophore(FIG. 20, panel B). Since T8 and T12 form similar single-nucleotideloops, 2Ap will be tested in these positions to design anultra-sensitive primer-probe of RT-QPA.

Development of Pteridine-Containing QPA Probes.

As described above, it is possible to incorporate labels other than 2Apinto the primer sequence. Thus, to design QPA probes for multiplextestes, similar studies will be performed using highly fluorescentpteridine analogs 3-methyl isoxanthopterin (3MI) (Ex348, Em431),6-methylisoxanthopterin (6MI) (Ex340, Em430) and(4-amino-6-methyl-8-(2¢-deoxy-â-D-ribofuranosyl)-7(8H)-pteridone (6AMP)(Ex330, Em435) with quantum yields of 0.88, 0.70 and 0.39 as monomerform, respectively. DNA containing pteridine probes is commerciallyavailable from Fidelity Systems. All three pteridine analogs showsignificant quenching upon incorporation into a DNA strand [Hawkins, M.E. (2008) Fluorescent pteridine probes for nucleic acid analysis.Methods in enzymology, 450, 201-231]. 6MI and 6AMP also show almostperfect base-pairing with cytosine and thymine, respectively. 3MI doesnot form stable base-pairs and its destabilization effect is similar toan effect of a single base-pair mismatch [Hawkins, M. E. (2008)Fluorescent pteridine probes for nucleic acid analysis. Methods inenzymology, 450, 201-231]. However, the analog will be studied due itshighest quantum yield.

Product Quantification Using QPA.

After finding optimal experimental conditions and primers, QPA will beused to quantify DNA products in real-time. The amplification will beperformed at various concentrations (from femtomolar to nanomolar) ofDNA template. QPA will be performed on DNA templates with incorporatedQPA-target sites obtained from the pUC18 plasmid (see Example 4).Initially, thermocycling QPA is planned. No product is expected atinitial temperature cycles as the fluorescence signal will be below thedetection threshold of the instrument (see FIG. 1). However, atthreshold cycles an increase in fluorescence is expected. Observing alinear dependence between log of the starting amount of templates andthe corresponding number of threshold cycles is also expected.Similarly, a linear dependence is expected upon isothermal QPA betweenlog of the starting amount of templates and time.

In order to compare RT-QPA with current detection mechanisms, amolecular beacon probe with Fluorescein tag will be designed. In theseexperiments, thermocycling QPA will be monitored by two independentways: 2Ap fluorescence at 370 nm (RT-QPA) and Fluorescein emission at521 nM (molecular beacon). Earlier detection of DNA product by 2Apsignal relative to the molecular beacon signal is expected.

The embodiments of the present invention recited herein are intended tobe merely exemplary and those skilled in the art will be able to makenumerous variations and modifications to it without departing from thespirit of the present invention. Notwithstanding the above, certainvariations and modifications, while producing less than optimal results,may still produce satisfactory results. All such variations andmodifications are intended to be within the scope of the presentinvention as defined by the claims appended hereto.

What is claimed is:
 1. A primer for amplification of a target nucleicacid, the primer comprising: a nucleic acid sequence, at least a portionof which comprises a G-rich sequence corresponding to the formulaG₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃, and wherein the G content of theG-rich sequence is equal to or greater than 70%; wherein the G-richsequence conforms into a quadruplex conformation during an extensionstep of an amplification process.
 2. The primer of claim 1, wherein theamplification process occurs at a substantially constant temperature. 3.The primer of claim 1, further including in its sequence at least onelabel chosen from 2Ap, 3MI, 6MI, and 6AMP.
 4. The primer of claim 1,wherein the G-rich sequence is chosen from SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:
 12. 5. A primer foramplification of a target nucleic acid, the primer comprising a firstsequence segment and a second sequence segment; wherein the firstsequence segment comprises a G-rich sequence corresponding to theformula G₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃, and wherein the G content ofthe G-rich sequence is equal to or greater than 70%; and wherein thesecond sequence segment is sufficiently complementary to the targetnucleic acid to hybridize therewith, and wherein the first sequencesegment does not hybridize to the second sequence segment or to asequence of the target nucleic acid, and wherein the G-rich sequence ofthe first sequence segment conforms into a quadruplex conformationduring an extension step of an amplification process.
 6. The primer ofclaim 5, wherein at least a portion of the first sequence segment has asequence comprising (GGGT)₄ [SEQ ID NO: 1].
 7. The primer of claim 5,further including in its sequence at least one label chosen from 2Ap,3MI, 6MI, and 6AMP.
 8. The primer of claim 7, wherein at least a portionof the first sequence segment has a sequence comprising SEQ ID NO: 10which is labeled as GGG(2Ap)GGGTGGGTGGG.
 9. The primer of claim 8,wherein the portion of the first sequence segment comprising 2Ap-G3T isattached to another sequence specific for use as a primer to detect adesired target nucleic acid sequence.
 10. The primer of claim 5, whereinthe portion of the first sequence segment having a sequence comprisingG₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃ has a sequence chosen from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 10 which is labeled as GGG(2Ap)GGGTGGGTGGG,SEQ ID NO: 11 which is labeled as GGG(2Ap)GGGTGGGTGG, and SEQ ID NO: 12which is labeled as GGG(2Ap)GGGTGGGTG.
 11. A process for amplifying atleast one target nucleic acid sequence contained in a nucleic acid or amixture of nucleic acids, the process comprising: treating a nucleicacid or a mixture of nucleic acids with at least one primer, wherein atleast a portion of the primer comprises a G-rich sequence correspondingto the formula G₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃, and wherein the Gcontent of the G-rich sequence is equal to or greater than 70%, whichallows the primer to conform into a quadruplex conformation during anextension step of an amplification process, under isothermic conditionssuch that, for the at least one nucleic acid sequence being amplified,an extension product of the at least one primer is synthesized which iscomplementary to a strand from the nucleic acid or a mixture of nucleicacids, wherein the at least one primer is selected so as to besufficiently complementary to the strand from the nucleic acid or amixture of nucleic acids to hybridize therewith such that the extensionproduct synthesized from the at least one primer, when it is separatedfrom its complement, can serve as a template for further synthesis of anextension product of another primer.
 12. The process of claim 11,further comprising separating the primer extension products from thetemplates on which they were synthesized to produce single-strandedmolecules.
 13. The process of claim 11, further comprising treating thesingle-stranded molecules with the at least one primer under isothermicconditions such that a primer extension product is synthesized usingeach of the single strands as a tem plate.
 14. The process of claim 11,wherein at least a portion of the primer sequence has a sequencecomprising (GGGT)₄ [SEQ ID NO:1].
 15. The process of claim 11, whereinthe at least one primer further includes in its sequence at least onelabel chosen from 2Ap, 3MI, 6MI, and 6AMP.
 16. The process of claim 15,wherein at least a portion of the primer sequence has a sequencecomprising SEQ ID NO: 10 which is labeled as GGG(2Ap)GGGTGGGTGGG. 17.The process of claim 16, wherein the portion of the primer sequencecomprising 2Ap-G3T is attached to another sequence specific for use as aprimer to detect a desired target nucleic acid sequence.
 18. The processof claim 11, wherein the portion of the primer comprising the G-richsequence corresponding to the formula G₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃has a sequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 10which is labeled as GGG(2Ap)GGGTGGGTGGG, SEQ ID NO: 11 which is labeledas GGG(2Ap)GGGTGGGTGG, and SEQ ID NO: 12 which is labeled asGGG(2Ap)GGGTGGGTG.
 19. A real-time quantification amplification methodfor detecting amplification of a target nucleic acid comprising:treating a nucleic acid or a mixture of nucleic acids with at least oneprimer, wherein at least a portion of the primer comprises a G-richsequence corresponding to the formula G₃₋₃₊N₁₋₇G₃₋₃₊N₁₋₇ G₃₋₃₊N₁₋₇G₀₋₃,and wherein the G content of the G-rich sequence is equal to or greaterthan 70%, which allows the primer to conform into a quadruplexconformation during an extension step of an amplification process, underconditions such that, for the at least one nucleic acid sequence beingamplified, an extension product of the at least one primer issynthesized which is complementary to a strand from the nucleic acid ora mixture of nucleic acids; wherein the at least one primer includes atleast one label that is quenched when the at least one primer is in anon-quadruplex conformation and that is detectable when the at least oneprimer is in a quadruplex conformation; and detecting the level of thelabel.
 20. The method of claim 19, wherein the label is chosen from 2Ap,3MI, 6MI, and 6AMP, and is incorporated into the at least one primer.21. A primer for amplification of a target nucleic acid, the primercomprising a first sequence segment and a second sequence segment;wherein at least a portion of the first sequence segment comprises asequence corresponding to the formula G₂₋₄N₁₋₃G₂₋₄N₁₋₃G₂₋₄N₁₋₃G₀₋₃,which allows the primer to conform into a quadruplex conformation duringan extension step of an amplification process; and wherein the secondsequence segment is sufficiently complementary to the target nucleicacid to hybridize therewith, and wherein the first sequence segment doesnot hybridize to the second sequence segment or to a sequence of thetarget nucleic acid.
 22. A primer for amplification of a target nucleicacid, the primer having a first sequence segment and a second sequencesegment; wherein at least a portion of the first sequence segment has asequence chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 10 which is labeled as GGG(2Ap)GGGTGGGTGGG, SEQ ID NO: 11 which islabeled as GGG(2Ap)GGGTGGGTGG, SEQ ID NO: 12 which is labeled asGGG(2Ap)GGGTGGGTG, SEQ ID NO: 20 which is labeled asGGTTGG(2Ap)GTGGTTGG, and SEQ ID NO: 24 which is labeled asGG(2Ap)TGGTGTGGTTGG, which allows the primer to conform into aquadruplex conformation during an extension step of an amplificationprocess; and wherein the second sequence segment is sufficientlycomplementary to the target nucleic acid to hybridize therewith, andwherein the first sequence segment does not hybridize to the secondsequence segment or to a sequence of the target nucleic acid.